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

Slides:

Advertisements

Similar presentations

Ane Y. Schmidt, Thomas v. O. Hansen, Lise B

Advertisements

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

Molecular Diagnostic Profiling of Lung Cancer Specimens with a Semiconductor-Based Massive Parallel Sequencing Approach Volker Endris, Roland Penzel,

Anna Sapino, Paul Roepman, Sabine C. Linn, Mireille H. J

Implementation of a Reliable Next-Generation Sequencing Strategy for Molecular Diagnosis of Dystrophinopathies Melissa Alame, Delphine Lacourt, Reda.

Droplet Digital PCR for Absolute Quantification of EML4-ALK Gene Rearrangement in Lung Adenocarcinoma Qiushi Wang, Xin Yang, Yong He, Qiang Ma, Li Lin,

Simultaneous Detection of Clinically Relevant Mutations and Amplifications for Routine Cancer Pathology Marlous Hoogstraat, John W.J. Hinrichs, Nicolle.

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.

Genotyping of DNA Samples Isolated from Formalin-Fixed Paraffin-Embedded Tissues Using Preamplification Renee Baak-Pablo, Vincent Dezentje, Henk-Jan.

Targeted Next-Generation Sequencing for Hereditary Cancer Syndromes

Emily M. Kudalkar, Naif A. M

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

Laila C. Schenkel, Charles Schwartz, Cindy Skinner, David I

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.

Analytical Validation of a Next-Generation Sequencing Assay to Monitor Immune Responses in Solid Tumors Jeffrey M. Conroy, Sarabjot Pabla, Sean T. Glenn,

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

Performance of Common Analysis Methods for Detecting Low-Frequency Single Nucleotide Variants in Targeted Next-Generation Sequence Data David H. Spencer,

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

False Positives in Multiplex PCR-Based Next-Generation Sequencing Have Unique Signatures Chad M. McCall, Stacy Mosier, Michele Thiess, Marija Debeljak,

Clinical Application of Picodroplet Digital PCR Technology for Rapid Detection of EGFR T790M in Next-Generation Sequencing Libraries and DNA from Limited.

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

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

Plasmid-Based Materials as Multiplex Quality Controls and Calibrators for Clinical Next- Generation Sequencing Assays David J. Sims, Robin D. Harrington,

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.

Simultaneous Detection of Clinically Relevant Mutations and Amplifications for Routine Cancer Pathology Marlous Hoogstraat, John W.J. Hinrichs, Nicolle.

Comparison of Allelic Discrimination by dHPLC, HRM, and TaqMan in the Detection of BRAF Mutation V600E Pablo Carbonell, María C. Turpin, Daniel Torres-Moreno,

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.

Utilization of Whole-Exome Next-Generation Sequencing Variant Read Frequency for Detection of Lesion-Specific, Somatic Loss of Heterozygosity in a Neurofibromatosis.

Christopher R. Cabanski, Vincent Magrini, Malachi Griffith, Obi L

Chromosomal Microarray Detection of Constitutional Copy Number Variation Using Saliva DNA Jennifer Reiner, Lisa Karger, Ninette Cohen, Lakshmi Mehta,

Donavan T. Cheng, Talia N. Mitchell, Ahmet Zehir, Ronak H

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

Martin Steinau, Sonya S. Patel, Elizabeth R. Unger

Jennifer Kerkhof, Laila C

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

Eric Samorodnitsky, Jharna Datta, Benjamin M

QuantiGene Plex Represents a Promising Diagnostic Tool for Cell-of-Origin Subtyping of Diffuse Large B-Cell Lymphoma John S. Hall, Suzanne Usher, Richard.

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

ColoSeq Provides Comprehensive Lynch and Polyposis Syndrome Mutational Analysis Using Massively Parallel Sequencing Colin C. Pritchard, Christina Smith,

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

SNPitty The Journal of Molecular Diagnostics

Cyclin E1 Is Amplified and Overexpressed in Osteosarcoma

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

Detection of Mutations in Myeloid Malignancies through Paired-Sample Analysis of Microdroplet-PCR Deep Sequencing Data Donavan T. Cheng, Janice Cheng,

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.

An Array-Based Analysis of MicroRNA Expression Comparing Matched Frozen and Formalin-Fixed Paraffin-Embedded Human Tissue Samples Xiao Zhang, Jiamin.

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

Analytical Validation and Application of a Targeted Next-Generation Sequencing Mutation-Detection Assay for Use in Treatment Assignment in the NCI-MPACT.

A Variant Detection Pipeline for Inherited Cardiomyopathy–Associated Genes Using Next-Generation Sequencing Théo G.M. Oliveira, Miguel Mitne-Neto, Louise.

Validation and Implementation of Targeted Capture and Sequencing for the Detection of Actionable Mutation, Copy Number Variation, and Gene Rearrangement.

Accurate Detection of Copy Number Changes in DNA Extracted from Formalin-Fixed, Paraffin-Embedded Melanoma Tissue Using Duplex Ratio Tests David A. Moore,

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

Comprehensive Genomic Profiling of Malignant Effusions in Patients with Metastatic Lung Adenocarcinoma Soo-Ryum Yang, Chieh-Yu Lin, Henning Stehr, Steven.

Gayatry Mohapatra, Rebecca A. Betensky, Ezra R

Validation for Clinical Use of, and Initial Clinical Experience with, a Novel Approach to Population-Based Carrier Screening using High-Throughput, Next-Generation.

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,

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

The History and Impact of Molecular Coding Changes on Coverage and Reimbursement of Molecular Diagnostic Tests Susan J. Hsiao, Mahesh M. Mansukhani,

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,

Preanalytic Variables and Tissue Stewardship for Reliable Next-Generation Sequencing (NGS) Clinical Analysis Paolo A. Ascierto, Carlo Bifulco, Giuseppe.

Presentation transcript:

Analytical Validation of Clinical Whole-Genome and Transcriptome Sequencing of Patient-Derived Tumors for Reporting Targetable Variants in Cancer Kazimierz O. Wrzeszczynski, Vanessa Felice, Avinash Abhyankar, Lukasz Kozon, Heather Geiger, Dina Manaa, Ferrah London, Dino Robinson, Xiaolan Fang, David Lin, Michelle F. Lamendola-Essel, Depinder Khaira, Esra Dikoglu, Anne-Katrin Emde, Nicolas Robine, Minita Shah, Kanika Arora, Olca Basturk, Umesh Bhanot, Alex Kentsis, Mahesh M. Mansukhani, Govind Bhagat, Vaidehi Jobanputra The Journal of Molecular Diagnostics Volume 20, Issue 6, Pages 822-835 (November 2018) DOI: 10.1016/j.jmoldx.2018.06.007 Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Positive predictive value (PPV) and sensitivity (SENS) for tumor/normal sequencing depth. Virtual tumor experiment PPV (solid line) and SENS (dotted line) percentages for single nucleotide variant callers MuTect (version 1.1.7) (black lines) and Strelka (version 1.0.14) (red lines) over a range of tumor/normal sequencing coverage. PPV increases with increased normal sequencing depth. SENS increases with increased tumor sequencing depth. The Journal of Molecular Diagnostics 2018 20, 822-835DOI: (10.1016/j.jmoldx.2018.06.007) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Virtual tumor variant allele frequency (VAF) and alternate allele read count versus read count (RC). A: VAF of true positive (TP; red dots) and false positive (FP; black dots) calls made by MuTect versus total RC at tumor/normal coverage (60×:30×, 80×:40×). B: Alternate allele read count of TP (red dots) and FP (black dots) calls made by MuTect (B) versus total RC at tumor/normal coverage (60×:30×, 80×:40×). The Journal of Molecular Diagnostics 2018 20, 822-835DOI: (10.1016/j.jmoldx.2018.06.007) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Whole-genome sequencing coverage. A: Representation of the coverage (mean and SD) for all variants in cancer census genes based on our validation sample data. Red filled circles indicate mean coverage of genes in our validation sample set. Black lines indicate SD limits from mean. Mean coverage per gene when targeting 80× is shown. B: Percentage of genome sequencing coverage for all 64 samples at 30× read depth (PCT_30X) and 40× read depth (PCT_40X). FF, fresh or frozen; FFPE, formalin-fixed, paraffin-embedded. The Journal of Molecular Diagnostics 2018 20, 822-835DOI: (10.1016/j.jmoldx.2018.06.007) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Limit of detection. A and B: Selected sample-specific variants of single nucleotide variant (SNV) and insertion/deletion (indel) (A) and copy number variant (CNV) (B) at 30%, 20%, and 10% tumor content in two samples, respectively. A: Variant allele frequencies in two samples [frozen and formalin-fixed, paraffin-embedded (FFPE)] with original tumor content of 91% and 65%, respectively, are shown with decreasing tumor content. B: Copy number log2(tumor/normal; T/N) of one focal amplification (EGFR, FFPE sample) and three deletions [two whole arm (1p, 19q, frozen sample) and one focal deletion (CDKN2A, FFPE sample)]. Inset shows 19q and 1p CNV log2(T/N) values at 30% to 10% tumor content for better resolution. The Journal of Molecular Diagnostics 2018 20, 822-835DOI: (10.1016/j.jmoldx.2018.06.007) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 DNA whole-genome sequencing (WGS) copy number correlation to genotyping chip copy number. The correlation of allele-specific copy number analysis of tumors (ASCAT) discrete copy number per gene to copy number per gene identified by DNA WGS is shown for 40 samples with tumor purity of 30%. The red squares represent the mean values in the distributions. The correlation of the mean values was r = 0.89, with total correlation at r = 0.70. The plot is shown with a y axis range limited to 0,30 copy number. T/N, tumor/normal. The Journal of Molecular Diagnostics 2018 20, 822-835DOI: (10.1016/j.jmoldx.2018.06.007) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S8 Copy number and B-allele frequency plots for tumor sample dilutions (frozen sample, CA-0061T). The frozen sample CA-0061 was diluted three times with its corresponding normal genomic DNA. Copy number log2 (tumor/normal; T/N) ratio and B-allele frequency across the genome are represented as called by NBIC-Seq for the original and dilution samples. A: Original tissue sample (90% tumor purity). B: Dilutions to 30% to 25% tumor content. C: 20% tumor content. D: 10% tumor content. The Journal of Molecular Diagnostics 2018 20, 822-835DOI: (10.1016/j.jmoldx.2018.06.007) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S9 Copy number and B-allele frequency plots for tumor sample dilutions (formalin-fixed, paraffin-embedded sample, G15-31T). The formalin-fixed, paraffin-embedded G15-35T was diluted three times with its corresponding normal genomic DNA. Copy number log2 (tumor/normal; T/N) ratio and B-allele frequency across the genome are represented as called by NBIC-Seq for the original and dilution samples. A: Original tissue sample (60% to 65% tumor purity). B: Dilutions to 30% tumor content. C: 20% to 15% tumor content. D: 10% tumor content. The Journal of Molecular Diagnostics 2018 20, 822-835DOI: (10.1016/j.jmoldx.2018.06.007) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S11 RNA sequencing coverage. A: Median coverage of 15 housekeeping genes plus two olfactory genes (ORA5A2 and OR11H4) used as control in all samples. B: Median coverage of 15 housekeeping genes from mRNA-prepared libraries. C: Median coverage of 15 housekeeping genes from total RNA-prepared libraries. Libraries prepared from mRNA samples exhibit greater coverage than libraries prepared from total RNA samples. D: A minimum library size of 40 million reads (vertical blue line) provides minimum median coverage of 30 reads for the housekeeping genes (horizontal red line), independent of input sample type. The Journal of Molecular Diagnostics 2018 20, 822-835DOI: (10.1016/j.jmoldx.2018.06.007) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions