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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

  • Drug development in oncology continues to experience high failure rate. We now understand that tumors are heterogeneous and dynamic, and we are seeing the importance of stratifying patients as well as continuously monitoring for resistant mutations. These measures may improve response rates to targeted therapies and their survival benefit.

    Next-generation sequencing (NGS) is emerging as a powerful clinical diagnostic tool expediting molecular stratification, and it is enhancing our knowledge of tumor clonal changes. This article presents feasibility of NGS in the context of targeted therapeutic approaches to cancer.

    “We are entering a promising time in cancer treatment, when the accumulated experience in cancer genome sequencing can be put to practical use in clinical practice,” says Shashikant Kulkarni, Ph.D., a professor of molecular and human genetics at Baylor College of Medicine, and a senior officer at Baylor Miraca Genetics Laboratories. “In just five years, NGS went from an experimental laboratory technology to clinically validated diagnostic tool.”

    Dr. Kulkarni points to the recent guidelines developed by the Next-Generation Sequencing: Standardization of Clinical Testing (Nex-StoCT) workgroup. Combined with more recent guidelines for bioinformatics, these important principles ensure that results from tests based on NGS are reliable and useful for clinical decisions.

    The workgroup was convened by the Centers for Disease Control (CDC), which expanded its genetic reference testing program to include reference standards for NGS characterization. Complementary efforts are being pursued by the National Institutes of Health and the National Institute for Standards and Technology (NIST).

    For example, NIST’s Genome in the Bottle team recently released the first DNA reference materials to gauge the performance of whole-genome and targeted panel sequencing tests. The development of these reference materials assists the FDA’s efforts to design a regulatory framework for NGS-based tests.

    The Nex-StoCT workgroup has proposed a three-stage validation process: platform, test-specific, and informatics pipeline validation. Such a process, notes Dr. Kulkarni, could increase confidence in the “critical knowledge gained via NGS.”

  • Shaping Cancer Clonal Evolution

    Dr. Kulkarni’s research is dedicated to the study of cancer clonal evolution and the analysis of the genetic changes that accumulate in the founder and side line clones. Dr. Kulkarni underscores critical knowledge gained through understanding of clonal selection and progression. Clonal evolution often leads to relapse of cancers such as acute myeloid leukemia (AML).

    NGS data suggests that initial clones escape chemotherapy and acquire additional DNA transversions likely due to DNA damage cause by chemotherapy itself. “If clonal evolution is shaped by chemotherapy,” emphasizes Dr. Kulkarni, “we should be focusing our efforts to enlist targeted therapy whenever possible.”

    Progress in personalized therapy, however, is still stymied by several major challenges. Correlation between mutations and phenotype is still poorly understood. ClinGen, a National Institutes of Health (NIH)-funded resource, is currently building an authoritative genomic knowledge database that defines the clinical relevance of genes and variants for use in cancer precision medicine, but these efforts are still in the early stages.

    Other challenges include lack of insurance reimbursement for NGS diagnostics, low awareness among clinicians, and a slow process of bringing targeted therapies to the market. “Change is slow,” complains Dr. Kulkarni. “But I am fortunate to be involved in changing the cancer treatment paradigm, one DNA base at a time. My dream is to democratize access to precision medicine to all cancer patients.”

  • Targeting Actionable Mutations

    The Center for Personalized Diagnostics (CPD) at the University of Pennsylvania aims to uncover actionable genetic mutations in individual cancers to allow more targeted and personalized treatment strategies. The Center’s core technology is NGS. The Center’s clinical director, Jennifer Morrissette, Ph.D., notes that the volume of analyses steadily grew from 50 cases per month at inception to reach the current volume of 250 sam>ples per month. The team zeroed in on a select panel of genes to create custom hematologic (68 genes) and solid tumor (47 genes) panels. “While our panels are not extensive, they are focused on targetable or prognostic genes,” adds Dr. Morrissette.

    Two case studies highlight the power of personalized therapies enabled by NGS. Both concern intrahepatic cholangiocarcinoma (ICC), which is known to be highly genetically diverse, with multiple translocations and mutations affecting over half of the genome.

    A stage IV patient of Arturo Loaiza-Bonilla, M.D., an assistant professor of clinical medicine at Penn, had undergone multiple rounds of standard-of-care therapies, only to develop new active metastasis. Genomic analysis reported a high-frequency mutation in the BRAF gene (pV600E), and the patient was deemed eligible for targeted BRAF/MEK inhibition therapy.

    The patient showed an extraordinary response, resolving all previous liver metastases. Dr. Morrissette mentions that the same mutation was since found in several other ICC patients, suggesting that it may be worth paying particular attention to V600E mutation in ICC. Of all cancers, ICC is especially genetically variable, and finding a targetable, if rare, mutation offers a promise to those patients who have it.

    In the second case, a multidisciplinary approach was particularly effective in determining the course of personalized therapy. The Abramson Cancer Center tumor board at Penn uses the genotype-to-phenotype concept to select targeted therapies or recommend a pertinent clinical trial to the patients. Martin Carroll’s group, using information from the CPD has been validating an Integrated Genetic Prognostic (IGP) model that combines cytogenetics with mutations in nine genes to predict a treatment course for younger patients with acute myeloid leukemia.

    “Being able to predict the course of disease informs clinicians about urgency of actions, for instance, to use targeted therapy, seek an immediate transplant, or to pursue palliative care,” explains Dr. Morrissette. “Our goal is to validate this model in a larger patient cohort.” The Center pursues other synergistic approaches, such as a combination of NGS and targeted single-gene testing to create an optimized and robust analysis workflow.

  • Finding Genomic Rearrangements

    Click Image To Enlarge +
    Researchers based at the Mayo Clinic’s Biomarker Discovery Program have combined laser capture microdissection and mate-pair sequencing to generate plots that detail large genomic alterations such as breakpoints, rearrangements, and copy-number variations. Such plots can help clinicians distinguish between independent primary tumors and metastasis. In this image, lines indicate bioinformatically associated breakpoints; line widths, numbers of associated mate-pair reads.

    “Genomic rearrangements represent a significant proportion of genetic abnormalities in cancer cells,” says George Vasmatzis, Ph.D., director of the Biomarker Discovery Program, Mayo Clinic. “Our program focused on this space early on and achieved significant advances in both sample preparation and informatics analysis.”

    Dr. Vasmatzis brought together a unique combination of technologies to identify large genomic rearrangements and translocations in liquid and solid tumors. The process begins with laser capture microdissection (LCM) to efficiently and accurately extract pure populations of tumor cells. The cells are applied directly into whole-genome amplification protocol, followed by mate-pair (MP) sequencing on Illumina instrumentation.

    In MP sequencing, the library preparation yields short inserts containing two adjacent fragments that were previously separated by 2–5 kB from each other in the genome. Combining data generated from MP library sequencing with that from short-insert paired-end reads provides a powerful ability to identify complex genomic rearrangements.

    “The MP approach is a rapid and economic way of sequencing the whole genome for such abnormalities,” asserts Dr. Vasmatzis. “Our Center has already completed over 1,500 tumor genomes.”

    Later this year, the Mayo Clinic plans to offer this test at their CLIA-certified lab. At first, it will support constitutional genetic analysis. Eventually, it will substitute for fluorescence in situ hybridization (FISH) and cytogenetic tests in the analysis of hematologic malignancies and solid tumors.

    Dr. Vasmatzis describes a typical clinical scenario where this technology may provide valuable information for clinicians: “It is critical to determine whether two distant nodules are genetically related. If they are related, it may indicate an aggressive late-stage tumor, but if they are not related, the disease may still be at stage I, making patient eligible for a potentially curable surgery.”

    Using just a few cells from the fine needle biopsy aspirations, the team is able to detect somatic translocations that are unique for each tumor clone. An added benefit is potential discovery of targetable rearrangements.

    This protocol may find a wider application in a nationwide screening of individuals with increased cancer risk, such as smokers. “It is still somewhat unclear how the trough of information that our test generates could be presented to physicians,” allows Dr. Vasmatzis. “We have been working on visualization strategies to make this complex information more readily understood and actionable.”

  • Fluid Cancer Monitoring

    Most patients with metastatic prostate cancer respond well to androgen deprivation therapy. However, many relapse and develop castration-resistant prostate cancer (CRPC).

    “Novel therapies targeting the androgen receptor (AR) demonstrate significant survival benefit,” says Delila Gasi-Tandefelt, Ph.D., Marie Curie Research Fellow, the Institute of Cancer Research (London). “However, 30% of patients do not respond at all, and among those who respond to therapy, invariably all develop resistance.”

    The Institute’s laboratory studies the origins and progression of treatment resistance. Molecular characterization of tumors taking serial biopsies could be impractical considering that most of CRPC metastasis develops in bones. In addition, heterogeneity of tumors renders comprehensive sampling rather challenging.

    Liquid biopsies present a minimally invasive way for longitudinal tracking of tumor molecular makeup. As part of the Institute’s treatment resistance team, Dr. Gasi-Tandefelt evaluated the liquid biopsy approach by conducting a comprehensive comparison of cancer tissues and circulating cell-free DNA from the same patients. She showed that tumor DNA found in blood is representative of the entire tumor burden.

    By following mutations in anchor genes, the team characterized the clonality of metastatic disease. The analysis suggested emergence of distinct mechanisms of resistance in different clones that arose or disappeared under treatment selection pressure. Sequential monitoring by liquid biopsies may ensure early discontinuation of therapies that drive resistance.

    “Our future research will further focus on evaluation of clonal response to targeted drug therapies,” informs Dr. Gasi-Tandefelt. “Tumors are very dynamic, especially under selective pressure. Thus, stratifying patients on the basis of past tumor biopsies is problematic.”

    NGS analysis of genetic AR aberrations in CRPC patients, before and after the start of abiraterone treatment, suggested the important role of somatic mutations and AR gain in resistance. The point mutations could be observed months before any clinical manifestation. This data is at the heart of a prospective clinical trial led by Gerhardt Attard, M.D., Ph.D., a senior researcher at the institute.

    At present, it appears that NGS could inform on the choice of treatment based on the type of the genomic aberration. The next frontier, Dr. Gasi-Tandefelt predicts, will be expansion from the targeted gene panels to whole-genome/whole-exome sequencing, garnering even more information for intelligent cancer treatments and single-cell analysis to enable detection of rare resistant clones.

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