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Aug 1, 2014 (Vol. 34, No. 14)

NGS Ready for Clinical Oncology Testing

From our August issue: Clinical genomics soon may be set ablaze by whole-exome and whole-genome sequencing.

  • Click Image To Enlarge +
    Exome sequencing has promise in oncology testing, particularly in the monitoring of disease progression and the prediction of therapeutic responses. To realize this promise, researchers are developing and validating assays of ever-wider scope. [Zmeel/iStock]

    Next-generation sequencing (NGS) not only continues to make steady advances in the molecular diagnosis of cancers, it also seems to fit perfectly with our current knowledge of the oncogenome.

    In particular, by making it possible to screen the entire coding sequence of cancer-related genes, NGS overcomes a key problem—cancer predisposition cannot be monitored by just a few hotspot mutations.

    The sensitivity, speed, and potentially decreased cost per sample make NGS a highly attractive technology. In fact, NGS may soon consolidate many other platforms. While some NGS applications, such as whole-genome sequencing, will probably have to wait several years before they spread and enter routine clinical use, other NGS applications are ready now.

    One NGS application that shows immediate promise for clinical oncology testing is exome sequencing. “We hope that in the near future, sequencing of the cancer exome will soon provide oncologists with information they need to identify and utilize treatment options based on the patient’s genomic profile,” says Helen Fernandes, Ph.D., associate professor of pathology and laboratory medicine, Weill Cornell Medical College. Dr. Fernandes, however, adds that NGS will become established in the clinic only after  certain challenges are addressed.

  • NGS Assays

    “We need to understand the role of several mutations in the pathogenesis and outcomes of tumors before the information can be used for patient care,” explains Dr. Fernandes. “For a start, we focus our efforts on a panel of 50 genes that could provide actionable information to our clinicians.”

    This panel, notes Dr. Fernandes, poses a practical difficulty. It must be used with an assay sensitive enough to identify a mutation present in less than 5% of a heterogenous population of tumor cells.

    The most common starting material typically comes from core biopsies embedded in paraffin for preservation. “We were able to successfully use this somewhat compromised material to amplify and analyze multiple genes simultaneously,” continues Dr. Fernandes. The test that has been validated in the clinical lab is able to use 10 ng of starting DNA to generate sequences covering 2,800 hotspots in the 50-gene panel.

    The power of this approach was demonstrated in a pilot study aiming to identify sequence variants in head and neck squamous cell carcinoma (HNSCC) with and without human papilloma virus (HPV). Surprisingly, tumors with increased HPV viral copy number are associated with better response to chemotherapy and prolonged survival.

    Dr. Fernandes’ team utilized NGS to systematically analyze somatic variants in virus-infected and virus-free samples. The data confirmed key differences in signaling pathways between the two cohorts. Notably, the HPV+ cells retained wild-type p53, which is known to promote cell apoptosis in response to chemotherapy.

    Several other genes identified in the study look promising as candidate targets for therapy. “In addition to significant insights in the pathogenesis of HNSCC we were also able to identify potential diagnostic markers that could become therapeutic targets,” asserts Dr. Fernandes. “NGS is rapidly becoming an invaluable tool in translational medicine and oncology.”

  • CLIA Certification

    Click Image To Enlarge +
    The Molecular Diagnostics Laboratory at the MD Anderson Cancer Center has taken steps to validate several NGS platforms, including the Life Technologies Ion Torrent PGM platform and the Illumina MiSeq platform. The laboratory rigorously tested several NGS-based protocols by analyzing and comparing the archival samples with known mutations and samples collected prospectively.

    “I believe that NGS has the potential to become a consolidated platform for multiple genotyping approaches,” says Rajyalakshmi Luthra, Ph.D., director of the Molecular Diagnostics Laboratory at the University of Texas MD Anderson Cancer Center. “NGS has capabilities not only to identify substitution mutations and insertions/deletions (indels), but also to find fusion transcripts and copy number changes.” Instead of managing multiple diagnostic platforms, a clinical diagnostic lab may rely on just one—a next-generation sequencer.

    Dr. Luthra points out that while the sequencing technology itself is already capable of detecting indels, variant-calling software packages have not yet reached desired consistency. “We continue visual inspection of raw reads until we can be sure that the software is capable of calling indels with high accuracy,” remarks Dr. Luthra.

    Nevertheless, the Molecular Diagnostics Laboratory has undertaken a concerted effort to validate several NGS platforms using CLIA (Clinical Laboratory Improvement Amendments) standards. For example, the Molecular Diagnostics Laboratory clinically validated the Ion Torrent PGM (Life Technologies) using 10 ng of DNA from 70 paraffin-embedded solid tumor samples. Variants detected by sequencing 740 mutational hotspots in 46 cancer-related genes in the panel were confirmed by orthogonal conventional platforms such as Sanger sequencing and pyrosequencing.

    Overall concordance between NGS and conventional platforms supported routine use of NGS in the CLIA-certified laboratory. The utility of this approach was elegantly applied to screening mutational hotspots in 54 cancer-related genes using MiSeq, Illumina’s NGS platform, as a part of diagnostic workup and treatment plan for patients with acute myeloid leukemia.

    “NGS provides for a single detection platform capable of analyzing multiple genes at the same time starting with a fraction of the material required in conventional multigene analysis,” notes Dr. Luthra.

    The laboratory rigorously tested several NGS-based protocols by analyzing and comparing the archival samples with known mutations and samples collected prospectively. NGS platforms showed high level of reproducibility and high detection sensitivity. This high sensitivity was useful in detecting the presence of low-level mutations that were missed by less sensitive conventional platforms during followup and relapse.

  • Universal Oncology Test System

    “As Dr. Luthra demonstrated in her study, MiSeq is highly applicable for molecular testing of oncogenes,” says Jennifer Stone, Ph.D., senior market manager for oncology, Illumina. The company has demonstrated conformity with the In Vitro Diagnostic Directive, and it has applied the CE mark to  the MiSeqDx™, making it the first and so far only sequencer cleared in Europe and by the FDA for clinical diagnostic applications.

    MiSeqDx will play an important role in the partnership forming between Illumina and the pharmaceutical industry. The goal of the partnership is to develop a universal oncology test system.

    “The current paradigm, [which emphasizes] single-analyte companion diagnostics, is changing,” continues Dr. Stone. “Tumor genetics is highly heterogeneous, and it changes over time with disease progression. The ability to monitor multiple analytes simultaneously and continuously would enable us to manage cancer as a chronic disease.”


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