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August 01, 2016 (Vol. 36, No. 14)

Sequencing Boosts Standard of Care

Clinical-Style Validation, Regulation, and Quality Control Are Spreading To NGS-Based Tests

  • Strengthening Quality Control

    “NGS is revolutionizing our approach to cancer therapeutics,” says Francine de Abreu, Ph.D., a genomic analyst at Dartmouth College. “It is essential for CLIA-certified laboratories to implement quality control (QC) programs to ensure accuracy and reproducibility of sequencing results.

    “At present, every CLIA laboratory has its own QC practices. We have established a comprehensive six-step QC process that ensures accurate sequencing results using formalin-fixed paraffin embedded (FFPE) tissues.”

    The workflow is based on the College of American Pathologists (CAP) framework developed over the past few years. It conceptualizes the overall NGS test process as composed of two major analytical components: a “wet bench” component and a “dry bench,” or bioinformatics component.

    The wet bench includes pre- and post-analytical checkpoints: DNA extraction, DNA quality, library preparation, and quantification. The dry bench includes bioinformatics steps: post-sequencing run, sample, and  variant metrics. For each run, the metrics verified are as follows: chip loading, usable sequences, polyclonality, and low-quality reads. Additional values that may be assessed for each individual sample include coverage uniformity and on-target reads.

    The complete NGS process can be monitored step by step. Dr. de Abreu explains that in addition to the mandatory routine testing of blinded CAP samples, many CLIA laboratories also agree to participate in additional proficiency testing through inter-laboratory sample exchanges. For its part, the Dartmouth-Hitchcock Medical Center has organized a Next-Generation Sequencing Project Team. It reviews and updates the accreditation checklist requirements specific to NGS to adapt them to meet the rapid evolution of NGS and its translation to clinical diagnostic testing.

    The Center’s laboratory for Clinical Genomics and Advanced Technology (CGAT) analyzed over 1,700 FFPE tumor tissues composed of multiple tumor types, with about 80% passing the “wet bench” QC checkpoints. A large percentage of identified somatic mutations were actionable.

    The Dartmouth-Hitchcock Medical Center established the Multidisciplinary Molecular Tumor Board to evaluate potential treatments for the actionable cases identified by the sequencing laboratory. In over 50% cases, the board was able to recommend therapy with a targeted agent. In a few patients treated with the recommended therapy, disease outcomes were positive, suggesting that increasing the awareness among patients and clinicians of the benefits of molecular testing could improve patient care.

  • Quantifying Cancer Biomarkers

    Click Image To Enlarge +
    The Bio-Rad QX200 Droplet Digital PCR system can provide absolute quantitation of target DNA or RNA molecules without the use of standard curves.

    The inherent sensitivity, rapid time to results, and cost effectiveness of droplet digital PCR (ddPCR) in the detection and quantification of DNA biomarkers identified by next-generation sequencing (NGS) makes ddPCR an attractive complement to NGS in the study
    and monitoring of cancer.

    One example of NGS and ddPCR working in tandem comes out of the laboratory of Lao Saal, M.D., Ph.D., assistant professor and head of the Translational Oncogenomics Unit at Lund University in Sweden. His team is studying whether levels of cell-free circulating tumor DNA, serially monitored from patients’ blood, can predict metastasis in early-stage cancer. His use of a liquid biopsy is less invasive than traditional, solid tumor biopsies, facilitating periodic measurement.

    Using low-coverage whole-genome NGS, Dr. Saal’s approach starts with identifying chromosomal rearrangements in patients’ primary tumors, which occur early in tumor development and are often shared among the tumor’s subclones. Although these rearrangements generally do not drive the cancer’s growth, they do serve as biomarkers for measuring tumor burden and are widely applicable to different cancer types.

    Dr. Saal’s team then uses
    Bio-Rad’s QX200 ddPCR system to monitor the quantity of the rearrangements in patients’ plasma samples using patient-specific ddPCR assays. With this method, the team was able to identify breast cancer recurrence in 86% of patients an average of 11 months (but as much as 3 years) prior to conventional clinical techniques.

    Dr. Saal’s laboratory also uncovered that biomarker levels are a predictor of metastasis and poor survival. Other research laboratories using similar methods have also illustrated the utility of digital PCR in liquid biopsy for monitoring disease load and treatment response in colorectal, rectal, gynecologic, and lung cancers.

    Dr. Saal is now commercializing this technique by forming SAGA Diagnostics, which offers liquid biopsy and companion diagnostic testing to healthcare organizations, biopharmaceutical companies, and academic institutions.

  • Detecting CNVs in Clinical Samples

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    Archer Universal DNA reagents are now integrated with the Archer VariantPlex system, which can generate target-specific libraries for next-generation sequencing.

    Several techniques have been employed to determine copy number, including array comparative genomic hybridization (aCGH) and quantitative PCR (qPCR).

    However, none of these methods are amenable to high-throughput, directed copy number variation (CNV) detection, according to a research team from the City of Hope National Medical Center and Archer DX. In a poster on detecting CNVs in clinical samples, Haimes et al. described the development of a directed next-generation sequencing (NGS)-based method to rapidly and quantitatively measure the copy number of tens and potentially hundreds of genes simultaneously.

    “This complete workflow, found in the Archer™ Universal DNA Kit, is powered by Anchored Multiplex PCR (AMP™) chemistry and processes dozens of samples in about six hours,” explained Josh Stahl, CSO and general manager. “By ligating a molecular barcode to randomly fragmented input DNA and then using AMP to simultaneously enrich for several regions of each target gene, we can accurately measure the relative copy number of each target gene in test samples by counting unique molecular barcodes associated with each target region.”

    The scientists validated their methodology with a 25-gene panel on a subset of NCI-60 cell lines by comparing their copy number measurements to those determined by both aCGH and qPCR. Results from both orthogonal methods strongly correlated with data from the team’s NGS-based method.

    “We multiplexed hundreds of samples on a single MiSeq® run and detected CNVs, both amplifications and deletions, of 2× magnitudes (and often lower) at extremely high confidence, indicating that this panel is amenable to highly multiplexed screens of potentially hundreds of samples,” added Stahl.

    “Furthermore, we demonstrated that our NGS-based CNV detection workflow and analysis is compatible with DNA extracted from formalin-fixed, paraffin-embedded (FFPE) samples, suggesting that this system could be adapted for use in clinical applications.”

  • Determining Allelic Frequencies of Human Cancers

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    The NEBNext Direct Cancer HotSpot Panel displays high uniformity of coverage across targets. Target bases were sequenced to at least 50%, 33%, and 25% of the mean read depth.

    Earlier this year, a team of scientists from Directed Genomics and New England Biolabs® (NEB®) presented a poster entitled “Application of target enrichment combined with unique molecular identifiers to determine allelic frequencies of human cancers” at the AACR conference in New Orleans. The study focused on the use of NEBNext Direct™, a target-enrichment technique developed for hybridization-based capture of genomic regions of interest.

    When this method is used, target genomic DNA sequences are isolated and converted into an Illumina-compatible library within seven hours. Unlike some alternative approaches that convert the entire genome into a library and select the targets as a final step, NEBNext Direct target-enrichment enzymatically removes off-target sequences and converts only target regions of the native DNA molecules into sequencer-ready libraries, according to Andrew Barry, target enrichment product marketing manager,  NEB.

    Target-enrichment and library-preparation strategies for next-generation sequencing typically utilize PCR amplification steps that introduce substantial bias across amplicons. They can also lead to the creation of duplicate sequence reads, which in turn affects the quantification accuracy of somatic allele frequencies, an important factor in understanding tumor progression.

    Unique molecular identifiers (UMIs) are molecular tags that label each molecule prior to library amplification with a 12-basepair randomized sequence incorporated into the the library adaptor. The UMI, notes Barry, allows sequence reads to be disambiguated as PCR duplicates, enabling an accurate assessment of variant allele frequencies and molecular copy number of the original sample.

    “In this study, we enriched control samples of known variant allele frequencies representing challenging sample types with the NEBNext Direct Cancer HotSpot Panel, and we used the incorporated UMIs to determine allelic frequencies of select cancer targets,” explains Barry. “This novel approach to target enrichment in conjunction with library preparation and the inclusion of UMIs enables filtering of PCR duplicate reads, offering improved sensitivity and more accurate assessment of  variant allele frequencies.”  

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