High-Throughput and Sensitive Quantification of Circulating Tumor DNA by Microfluidic- Based Multiplex PCR and Next-Generation Sequencing Yinghui Guan,

Slides:

Advertisements

Similar presentations

Clinical Laboratory Analysis of Immunoglobulin Heavy Chain Variable Region Genes for Chronic Lymphocytic Leukemia Prognosis Philippe Szankasi, David.

Advertisements

Martina I. Lefterova, Peidong Shen, Justin I

Comparison of Clinical Targeted Next-Generation Sequence Data from Formalin-Fixed and Fresh-Frozen Tissue Specimens David H. Spencer, Jennifer K. Sehn,

A Highly Sensitive and Quantitative Test Platform for Detection of NSCLC EGFR Mutations in Urine and Plasma Karen L. Reckamp, MD, Vladislava O. Melnikova,

Carol Beadling, Tanaya L. Neff, Michael C

A Targeted High-Throughput Next-Generation Sequencing Panel for Clinical Screening of Mutations, Gene Amplifications, and Fusions in Solid Tumors Rajyalakshmi.

Assessing Copy Number Alterations in Targeted, Amplicon-Based Next-Generation Sequencing Data Catherine Grasso, Timothy Butler, Katherine Rhodes, Michael.

Martina I. Lefterova, Peidong Shen, Justin I

One-Step Ligation on RNA Amplification for the Detection of Point Mutations Lei Zhang, Jingjing Wang, Mia Coetzer, Stephanie Angione, Rami Kantor, Anubhav.

Emily M. Kudalkar, Naif A. M

Validation of a Clinical-Grade Assay to Measure Donor-Derived Cell-Free DNA in Solid Organ Transplant Recipients Marica Grskovic, David J. Hiller, Lane.

Single-Color Digital PCR Provides High-Performance Detection of Cancer Mutations from Circulating DNA Christina Wood-Bouwens, Billy T. Lau, Christine.

Comprehensive Screening of Gene Copy Number Aberrations in Formalin-Fixed, Paraffin-Embedded Solid Tumors Using Molecular Inversion Probe–Based Single-

A Method to Evaluate the Quality of Clinical Gene-Panel Sequencing Data for Single- Nucleotide Variant Detection Chung Lee, Joon S. Bae, Gyu H. Ryu, Nayoung.

Optimization of a Relative Telomere Length Assay by Monochromatic Multiplex Real- Time Quantitative PCR on the LightCycler 480 Anthony Y.Y. Hsieh, Sara.

Methodologic Considerations in the Application of Next-Generation Sequencing of Human TRB Repertoires for Clinical Use Liwen Xu, Xiaoqing You, PingPing.

Hendrikus J. Dubbink, Peggy N. Atmodimedjo, Ronald van Marion, Niels M

A Quantitative Allele-Specific PCR Test for the BRAF V600E Mutation Using a Single Heterozygous Control Plasmid for Quantitation Philippe Szankasi, N.

Somatic Mutation Screening Using Archival Formalin-Fixed, Paraffin-Embedded Tissues by Fluidigm Multiplex PCR and Illumina Sequencing Ming Wang, Leire.

Targeted, High-Depth, Next-Generation Sequencing of Cancer Genes in Formalin- Fixed, Paraffin-Embedded and Fine-Needle Aspiration Tumor Specimens Andrew.

MethySYBR, a Novel Quantitative PCR Assay for the Dual Analysis of DNA Methylation and CpG Methylation Density Pang-Kuo Lo, Hanano Watanabe, Pi-Chun.

Robustness of Amplicon Deep Sequencing Underlines Its Utility in Clinical Applications Vera Grossmann, Andreas Roller, Hans-Ulrich Klein, Sandra Weissmann,

Multiplex Preamplification of Serum DNA to Facilitate Reliable Detection of Extremely Rare Cancer Mutations in Circulating DNA by Digital PCR Jennifer.

Elena Castellanos-Rizaldos, Cloud Paweletz, Chen Song, Geoffrey R

Molecular Analysis of Circulating Cell-Free DNA from Lung Cancer Patients in Routine Laboratory Practice Stephan Bartels, Sascha Persing, Britta Hasemeier,

Validation of a Clinical-Grade Assay to Measure Donor-Derived Cell-Free DNA in Solid Organ Transplant Recipients Marica Grskovic, David J. Hiller, Lane.

Application of Single-Molecule Amplification and Resequencing Technology for Broad Surveillance of Plasma Mutations in Patients with Advanced Lung Adenocarcinoma

Angela Leo, Andrew M. Walker, Matthew S

Clinical Validation of a Next-Generation Sequencing Genomic Oncology Panel via Cross-Platform Benchmarking against Established Amplicon Sequencing Assays

Application of Single-Molecule Amplification and Resequencing Technology for Broad Surveillance of Plasma Mutations in Patients with Advanced Lung Adenocarcinoma

Quantitative Expression Profiling in Formalin-Fixed Paraffin-Embedded Samples by Affymetrix Microarrays Diana Abdueva, Michele Wing, Betty Schaub, Timothy.

A Method for Next-Generation Sequencing of Paired Diagnostic and Remission Samples to Detect Mitochondrial DNA Mutations Associated with Leukemia Ilaria.

Noninvasive Molecular Monitoring in Multiple Myeloma Patients Using Cell-Free Tumor DNA Giulia Biancon, Silvia Gimondi, Antonio Vendramin, Cristiana.

Clinical Relevance of Sensitive and Quantitative STAT3 Mutation Analysis Using Next- Generation Sequencing in T-Cell Large Granular Lymphocytic Leukemia

Molecular Diagnosis of Autosomal Dominant Polycystic Kidney Disease Using Next- Generation Sequencing Adrian Y. Tan, Alber Michaeel, Genyan Liu, Olivier.

Copy Number Variation Sequencing for Comprehensive Diagnosis of Chromosome Disease Syndromes Desheng Liang, Ying Peng, Weigang Lv, Linbei Deng, Yanghui.

Evaluation of the Cepheid GeneXpert BCR-ABL Assay

Accurate Quantification of T Cells by Measuring Loss of Germline T-Cell Receptor Loci with Generic Single Duplex Droplet Digital PCR Assays Willem H.

Karin D. Berg, Cynthia L. Glaser, Richard E. Thompson, Stanley R

Study of Preanalytic and Analytic Variables for Clinical Next-Generation Sequencing of Circulating Cell-Free Nucleic Acid Meenakshi Mehrotra, Rajesh.

Quantification of the Mutant CALR Allelic Burden by Digital PCR

A Rapid and Sensitive Next-Generation Sequencing Method to Detect RB1 Mutations Improves Care for Retinoblastoma Patients and Their Families Wenhui L.

Clinical Laboratory Analysis of Immunoglobulin Heavy Chain Variable Region Genes for Chronic Lymphocytic Leukemia Prognosis Philippe Szankasi, David.

Catherine E. Cottrell, Hussam Al-Kateb, Andrew J. Bredemeyer, Eric J

Effects of Collection and Processing Procedures on Plasma Circulating Cell-Free DNA from Cancer Patients Bente Risberg, Dana W.Y. Tsui, Heather Biggs,

Validation and Implementation of a Custom Next-Generation Sequencing Clinical Assay for Hematologic Malignancies Michael J. Kluk, R. Coleman Lindsley,

Study of Preanalytic and Analytic Variables for Clinical Next-Generation Sequencing of Circulating Cell-Free Nucleic Acid Meenakshi Mehrotra, Rajesh.

Analytical Validation of Clinical Whole-Genome and Transcriptome Sequencing of Patient-Derived Tumors for Reporting Targetable Variants in Cancer Kazimierz.

Comparison of Next-Generation Sequencing Panels and Platforms for Detection and Verification of Somatic Tumor Variants for Clinical Diagnostics Maksym.

Olivier Gruselle, Thierry Coche, Jamila Louahed

Development and Validation of a Template-Independent Next-Generation Sequencing Assay for Detecting Low-Level Resistance-Associated Variants of Hepatitis.

Technical Validation of a Next-Generation Sequencing Assay for Detecting Clinically Relevant Levels of Breast Cancer–Related Single-Nucleotide Variants.

Deamination Effects in Formalin-Fixed, Paraffin-Embedded Tissue Samples in the Era of Precision Medicine Seokhwi Kim, Charny Park, Yongick Ji, Deok G.

Figure 1. Plasma next-generation sequencing (NGS) assay workflow, comparison of variant allelic fraction with ... Figure 1. Plasma next-generation sequencing.

Mutation Analysis of SLC26A4 for Pendred Syndrome and Nonsyndromic Hearing Loss by High-Resolution Melting Neng Chen, Lisbeth Tranebjærg, Nanna Dahl.

A Clinically Validated Diagnostic Second-Generation Sequencing Assay for Detection of Hereditary BRCA1 and BRCA2 Mutations Ian E. Bosdet, T. Roderick.

Evaluation of the Abbott Investigational Use Only RealTime HIV-1 Assay and Comparison to the Roche Amplicor HIV-1 Monitor Test, Version 1.5 Michael T.

Amplification Refractory Mutation System, a Highly Sensitive and Simple Polymerase Chain Reaction Assay, for the Detection of JAK2 V617F Mutation in Chronic.

Evaluation of Second-Generation Sequencing of 19 Dilated Cardiomyopathy Genes for Clinical Applications Sivakumar Gowrisankar, Jordan P. Lerner-Ellis,

A Platform for Rapid Detection of Multiple Oncogenic Mutations With Relevance to Targeted Therapy in Non–Small-Cell Lung Cancer Zengliu Su, Dora Dias-Santagata,

Clinical Validation of a Next-Generation Sequencing Screen for Mutational Hotspots in 46 Cancer-Related Genes Rajesh R. Singh, Keyur P. Patel, Mark J.

Custom Design of a GeXP Multiplexed Assay Used to Assess Expression Profiles of Inflammatory Gene Targets in Normal Colon, Polyp, and Tumor Tissue Janice.

Detection of BRCA1 and BRCA2 Ashkenazi Jewish Founder Mutations in Formalin- Fixed Paraffin-Embedded Tissues Using Conventional PCR and Heteroduplex/Amplicon.

Nathan D. Montgomery, Sara R. Selitsky, Nirali M. Patel, D

Improved Detection of the KIT D816V Mutation in Patients with Systemic Mastocytosis Using a Quantitative and Highly Sensitive Real-Time qPCR Assay Thomas.

HPV Genotype Detection Using Hybrid Capture Sample Preparation Combined with Whole Genome Amplification and Multiplex Detection with Luminex XMAP Brian.

Multilaboratory Assessment of a New Reference Material for Quality Assurance of Cell- Free Tumor DNA Measurements Hua-Jun He, Erica V. Stein, Yves Konigshofer,

Analytical Validation of a Highly Sensitive, Multiplexed Chronic Myeloid Leukemia Monitoring System Targeting BCR-ABL1 RNA Justin T. Brown, Ion J. Beldorth,

Development of a Novel Next-Generation Sequencing Assay for Carrier Screening in Old Order Amish and Mennonite Populations of Pennsylvania Erin L. Crowgey,

MammaPrint and BluePrint Molecular Diagnostics Using Targeted RNA Next-Generation Sequencing Technology Lorenza Mittempergher, Leonie J.M.J. Delahaye,

Presentation transcript:

High-Throughput and Sensitive Quantification of Circulating Tumor DNA by Microfluidic- Based Multiplex PCR and Next-Generation Sequencing Yinghui Guan, Oleg Mayba, Thomas Sandmann, Shan Lu, Younjeong Choi, Walter C. Darbonne, Vincent Leveque, Lisa Ryner, Eric Humke, Nga W.R. Tam, Sundari Sujathasarma, Anna Cheung, Richard Bourgon, Mark R. Lackner, Yulei Wang The Journal of Molecular Diagnostics Volume 19, Issue 6, Pages 921-932 (November 2017) DOI: 10.1016/j.jmoldx.2017.08.001 Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 PreAmp MMP-Seq work flow for ctDNA. This is a schematic illustration showing a work flow for a ctDNA panel. Plasma DNA (5 ng) is first preamplified with 96 to 121 multiplex PCR assays, followed by multiplex target enrichment in Fluidigm Access Array, as described previously.19 The libraries were then sequenced on a MiSeq instrument. BC, barcode sequence; cfDNA, cell-free DNA; CS, Fluidigm common sequence; F, forward primer; PE, Illumina sequencing paired-end sequence; R, reverse primer; TS, target specific. The Journal of Molecular Diagnostics 2017 19, 921-932DOI: (10.1016/j.jmoldx.2017.08.001) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Performance assessment of PreAmp MMP-Seq work flow. A: Latin square (LSQ) control DNA samples are run with the PreAmp and regular MMP-Seq work flows on a ctDNA panel to evaluate amplicon coverage. B: Expected mutations from LSQ samples detected with no PreAmp and PreAmp MMP-Seq protocols are plotted to compare sensitivities of the two protocols. C: Allele fractions (AFs) of variant calls from two replicate PreAmp MMP-Seq runs are compared to evaluate reproducibility. The dashed lines in B and C mark the 0 point of AFs on the x and y axes. D: Linearity of four expected mutations in LSQ samples detected by the PreAmp protocol. Solid lines represent linear regression trend line, and dotted lines on each side represent 95% CIs. E: There are 142 engineered hotspot mutations in the AcroMetrix Oncology Hotspot Control (AOHC) that are covered by ctDNA panel amplicons. The distributions of allele frequencies of the 142 hotspots are plotted in histograms. Separate panels represent data from different titrations of AOHC samples. The expected average frequency measured independently by the vendor through next-generation sequencing is indicated on top of each panel of histograms. The x axis is shown on a log2 scale. The expected AFs of serially diluted AOHC samples are highlighted with dashed lines in each histogram. The Journal of Molecular Diagnostics 2017 19, 921-932DOI: (10.1016/j.jmoldx.2017.08.001) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Determination of position/substitution-specific errors with the empirical bayesian error model. Forty-three normal plasma samples were profiled with the ctDNA panel using the PreAmp MMP-Seq. A: Error rate distribution of all ctDNA amplicons. B: Error rate distribution of ctDNA amplicons labeled by reference (Ref) bases. C: Error distribution of amplicon HRAS p.Q61H_1 (Supplemental Table S2) separated by all potential base substitution types. Only nonzero observed error rates are used for visualization. D: Density plot of estimated error distribution, as shown in a black line, and observed error rates (red ticks at the bottom) are shown for two representative positions within the ctDNA panel of amplicons. Chr, chromosome; MAD, median absolute deviation. The Journal of Molecular Diagnostics 2017 19, 921-932DOI: (10.1016/j.jmoldx.2017.08.001) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Amplicon coverage and error rates of tissue hotspots in normal plasma. Longitudinal plasma samples from φ DMUC5754A trials were profiled using the ctDNA PreAmp MMP-Seq. A: Average sequencing coverage per amplicon in 81 plasma samples. B: Error rates and their 95% CIs (error bars) calculated on the basis of an empirical bayesian model are plotted for 29 positions where clinically relevant variants were detected in matched formalin-fixed, paraffin-embedded tissues. Solid lines indicate the average, and the dashed line in B shows the median. The Journal of Molecular Diagnostics 2017 19, 921-932DOI: (10.1016/j.jmoldx.2017.08.001) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Clinical utilities of ctDNA work flow on monitoring disease burden. Longitudinal levels of mutations found in both plasma and matched tissues from four patients are shown. The ctDNA levels (pg/mL plasma) were calculated by multiplying the frequency of a mutation with the mass (pg) of cell-free DNA yield/mL plasma. The levels of ctDNA at certain treatment time points are compared with those at baseline before treatment. The y axes of all four subplots are percentage change from baseline. Percentage change in longitudinal HE4 or CA-19-9 levels and tumor size are also plotted in each graph for comparison. Also, 40% HE4 and 50% CA-19-9 reductions are associated with clinical response in DMUC5754A and are shown in a dashed line for each patient. SLD, size at the longest dimension. The Journal of Molecular Diagnostics 2017 19, 921-932DOI: (10.1016/j.jmoldx.2017.08.001) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions