Liangjing Chen

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

Implementation of highly sophisticated technologies, such as next-generation sequencing (NGS), into routine clinical practice requires compatibility with common tumor biopsy types, such as formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration specimens, and validation metrics for platforms, controls, and data analysis pipelines. In this study, a two-step PCR enrichment workflow was used to assess 540 known cancer-relevant variants in 16 oncogenes for high-depth sequencing in tumor samples on either mature (Illumina GAIIx) or emerging (Ion Torrent PGM) NGS platforms. The results revealed that the background noise of variant detection was elevated approximately twofold in FFPE compared with cell line DNA. Bioinformatic algorithms were optimized to accommodate this background. Variant calls from 38 residual clinical colorectal cancer FFPE specimens and 10 thyroid fine-needle aspiration specimens were compared across multiple cancer genes, resulting in an accuracy of 96.1% (95% CI, 96.1% to 99.3%) compared with Sanger sequencing, and 99.6% (95% CI, 97.9% to 99.9%) compared with an alternative method with an analytical sensitivity of 1% mutation detection. A total of 45 of 48 samples were concordant between NGS platforms across all matched regions, with the three discordant calls each represented at <10% of reads. Consequently, NGS of targeted oncogenes in real-life tumor specimens using distinct platforms addresses unmet needs for unbiased and highly sensitive mutation detection and can accelerate both basic and clinical cancer research.

An Information-Rich CGG Repeat Primed PCR That Detects the Full Range of Fragile X Expanded Alleles and Minimizes the Need for Southern Blot Analysis

(CGG)(n) repeat expansion in the FMR1 gene is associated with fragile X syndrome and other disorders. Current methods for FMR1 molecular testing rely on Southern blot analysis to detect expanded alleles too large to be PCR-amplified and to identify female homozygous alleles that often confound interpretations of PCR data. A novel, single-tube CGG repeat primed FMR1 PCR technology was designed with two gene-specific primers that flank the triplet repeat region, as well as a third primer that is complementary to the (CGG)(n) repeat. This PCR was evaluated with 171 unique DNA samples, including a blinded set of 146 clinical specimens. The method detected all alleles reported by Southern blot analysis, including full mutations in 66 clinical samples and comprised up to 1300 CGG. Furthermore, a blinded cohort of 42 female homozygous and heterozygous specimens, including 21 with full mutation alleles, was resolved with 100% accuracy. Last, AGG interrupter sequences, which may influence the risk of (CGG)(n) expansion in the children of some carriers, were each correctly identified in 14 male and female clinical samples as referenced to DNA sequencing. As a result, this PCR provides robust detection of expanded alleles and resolves allele zygosity, thus minimizing the number of samples that require Southern blot analysis and producing more comprehensive FMR1 genotyping data than other methods.

High-resolution methylation polymerase chain reaction for fragile X analysis: Evidence for novel FMR1 methylation patterns undetected in Southern blot analyses

Purpose: Fragile X syndrome is associated with the expansion of CGG trinucleotide repeats and subsequent methylation of the FMR1 gene. Molecular diagnosis of fragile X currently requires Southern blot analysis to assess methylation. This study describes the evaluation of a polymerase chain reaction-only workflow for the determination of methylation status across a broad range of FMR1 genotypes in male and female specimens.Methods: We evaluated a novel method that combines allele-specific methylation polymerase chain reaction and capillary electrophoresis with eight cell line and 80 clinical samples, including 39 full mutations. Methylation status was determined using a three-step workflow: (1) differential treatment of genomic DNA using a methylation-sensitive restriction enzyme; (2) polymerase chain reaction with two sets of dye-tagged primers; and (3) amplicon sizing by capillary electrophoresis. All samples were analyzed by both methylation polymerase chain reaction and Southern blot analysis.Results: FMR1 methylation status and CGG repeat sizing were accurately and reproducibly determined in a set of methylation controls and genomic DNA samples representing a spectrum of CGG repeat lengths and methylation states. Moreover, methylation polymerase chain reaction revealed allele-specific methylation patterns in premutation alleles that were unobtainable using Southern blot analysis.Conclusions: Methylation polymerase chain reaction enabled high throughput, high resolution, and semiquantitative methylation assessments of FMR1 alleles, as well as determinations of CGG repeat length. Results for all samples were concordant with corresponding Southern blot analyses. As a result, this study presents a polymerase chain reaction-based method for comprehensive FMR1 analysis. In addition, the identification of novel methylation mosaic patterns revealed after polymerase chain reaction and capillary electrophoresis may be relevant to several FMR1 disorders.

Functional DNA quantification guides accurate next-generation sequencing mutation detection in formalin-fixed, paraffin-embedded tumor biopsies

The formalin-fixed, paraffin-embedded (FFPE) biopsy is a challenging sample for molecular assays such as targeted next-generation sequencing (NGS). We compared three methods for FFPE DNA quantification, including a novel PCR assay (‘QFI-PCR’) that measures the absolute copy number of amplifiable DNA, across 165 residual clinical specimens. The results reveal the limitations of commonly used approaches, and demonstrate the value of an integrated workflow using QFI-PCR to improve the accuracy of NGS mutation detection and guide changes in input that can rescue low quality FFPE DNA. These findings address a growing need for improved quality measures in NGS-based patient testing.

Abstract 5574: A comprehensive, targeted next-generation sequencing method that rapidly and accurately detects circulating tumor DNA variants at 0.1% frequency in plasma samples

Mutation analysis of circulating tumor DNA (ctDNA) in blood-based liquid biopsies provides a minimally invasive approach to detect and monitor disease. Existing next-generation sequencing (NGS) liquid biopsy techniques have laborious and/or inefficient workflows, heuristic error-correction algorithms, and variable performance with clinical tumor-plasma samples. We present a method that combines an efficient wet-bench workflow with accurate drybench analytics to reduce costs and turnaround time and is relevant to clinical research and patient testing.

Abstract 1389: A unified and streamlined targeted sequencing system for the quantification of DNA mutations and RNA expression markers in lung cancer

Introduction: The promise of precision medicine relies on the identification of DNA and RNA markers that can individualize patient management. Methods such as next-generation sequencing (NGS) can deduce DNA or RNA sequences, but both types of nucleic acid have not been efficiently and effectively combined into a single NGS workflow. We describe a comprehensive methodology for targeted clinical NGS that reports DNA and RNA variants, provides a streamlined workflow, and accommodates low-input total nucleic acid (TNA) from challenging clinical specimens. Methods: Sample QC was performed using a novel qPCR assay that quantifies discrete populations of amplifiable DNA and RNA from TNA material. PCR-based target enrichment was performed using QuantideX® NGS reagents (Asuragen) and sequenced on the MiSeq® System (Illumina). Bioinformatic analyses were conducted using QuantideX® Reporter (Asuragen), a software suite that directly incorporates pre-analytical QC information into the variant calling. Results: Targeted DNA- and RNA-seq panels were developed that query 54 lung cancer DNA targets and 135 RNA targets, including >100 gene fusions and mRNA expression markers associated with clinical actionability. Gene-specific primers were formulated as multiplex PCR or RT-PCR reactions to independently interrogate DNA or RNA variants, respectively, from TNA or combined into a 189-plex RT-PCR to report multi-categorical nucleic acid variants. Integration of the bioinformatics pipeline and variant caller with wet-lab QC results enhanced mutation call sensitivity at The NGS panels were assessed with cell-line and synthetic controls and a residual clinical cohort of 97 NSCLC FFPE specimens. Mutations were accurately detected from as few as 5-10 DNA templates and down to Conclusions: QuantideX targeted NGS chemistries can unify the analysis of DNA and RNA markers associated with lung cancer and report SNVs, indels, fusions, and aberrantly expressed mRNA transcripts from a single NGS run. This novel technology offers reliable, accurate and comprehensive molecular characterizations of challenging tumor specimens by integrating wet-bench and dry-bench methods and improving the efficacy of routine laboratory testing. Citation Format: Gary J. Latham, Julie Krosting, Michael Dodge, Robert Zeigler, Liangjing Chen, Jason Plyler, Shobha Gokul, Junya Fujimoto, Vassiliki Papadimitrakopoulou, Ignacio Wistuba, Richard Blidner, Brian Haynes. A unified and streamlined targeted sequencing system for the quantification of DNA mutations and RNA expression markers in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1389.