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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.

  • Illumina has already established itself as a leader in supplying NGS-based oncology tests for clinical research, with anywhere between 26 and 94 genes in the panels. A universal oncology test system would incorporate 60–70 actionable cancer genes of specific interest to the pharmaceutical partners.

    Many analytes could represent targets of new drugs in the pharmaceutical pipeline. Illumina envisions that such a universal platform would be able to aggregate and systematize results from both academic and drug development efforts. Wide distribution of a universal test would enable broad patient access.

    Dr. Stone emphasizes that NGS seems to be a nearly ideal solution for oncology diagnostics because of its ability to profile multiple genes using only a small amount of starting material and to generate results within a short period of time. Continuously collected data would help to monitor the tumor genetic landscape and adjust therapies as needed to keep tumors from evolving.

    “The inherent genetic complexity of cancer and the diversity of the pharmaceutical pipeline make it a challenge to pair drugs with patients who may benefit from them,” observes Dr. Stone. “Our multiplexed tool would enable better pairing at both clinical trial and treatment stages.”

    Illumina plans to deliver a fully regulatory-compliant solution that will include a universal test; the MiSeqDx; and sample extraction, library prep, and other supporting technologies.

  • Actionable Physician Reports

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    To develop a system capable of generating actionable sequencing reports for oncologists, researchers at the Mt. Sinai School of Medicine are leveraging predictive network models that can comb through vast amounts of publicly available knowledge and integrate information at the DNA, RNA, and protein levels. The researchers also build a decision tree for each FDA-approved treatment as well as drugs that have not yet been approved but appear to have potential benefits.

    “Our goal is to develop actionable sequencing reports for physicians,” says Rong Chen, Ph.D., assistant professor and director of clinical genome sequencing, Icahn School of Medicine at Mount Sinai. “These reports indicate which drugs would most likely benefit a particular patient, which drugs would have toxicities, and which clinical trials might enroll the patient.”

    Dr. Chen’s team builds a decision tree for each FDA-approved treatment as well as drugs that have not yet been approved but appear to have potential benefits, as indicated by molecular network information or in vitro or in vivo studies. A sophisticated Hadoop-based search engine combs though vast amounts of publicly available knowledge and integrates information at the DNA, RNA, and protein levels.

    “While the bioinformatics workflow to generate driver mutation analysis is very involved, the resulting report should be tailored to the physician’s workflow,” continues Dr. Chen. “We received a lot of feedback from oncologists on how to provide the most useful information and layouts. In the future, we hope to automate the process to reach a critical mass of physicians and then start receiving their feedback regarding success of the suggested treatment.”

    Dr. Chen recognizes that genome interpretation may be confounded by inaccuracies that arise from the sequencing process itself or related procedures such as alignment, variant calling, and functional annotation. Dr. Chen’s team deploys multiple strategies to address the known causes of error. These include sequencing on two different platforms and utilizing multiple variant callers and sequence aligners. In addition, the team manually reviews the most important calls and checks raw reads of known driver mutations to avoid false-negative calls. “Mistakes are unacceptable,” insists Dr. Chen. “Treatment outcomes depend on accurate information.”

    Dr. Chen’s group continues to develop methods to integrate and translate various molecular measurements in the public repositories into biomarkers for the diagnosis of disease. For example, the group’s ActiVar database contains 110 million distinct variants; 600,000 genome, exome, and genotyping data points; and manually curated human genetics papers.

    Dr. Chen’s latest initiative, the Resiliance Project, aims to find secondary mutations that “balance” the disease-causing mutations. It aims to find individuals with catastrophic genetic mutations that somehow remain healthy, presumably via protections afforded by yet undiscovered genetic or environmental factors.

    The Resiliance Project is the first systematic attempt to recruit “unexpected heroes” —people willing to donate their DNA in order to further research into mechanisms of disease prevention. “This project takes full advantage of all our previous experience in generating the highest quality sequencing and annotation workflow,” concludes Dr. Chen.

  • Overlooked Oncogenes

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    In a case of thyroid cancer that was evaluated at the Holycross Cancer Center in Poland, Sanger sequencing did not reveal mutations in the BRAF (V600E) gene. Instead, an NGS panel was used to uncover oncogenic mutations in a different gene (NRAS). This gene has a positive predictive value for malignancy of about 80%.

    Next-generation sequencing has a bright future,” comments Artur Kowalik, Ph.D., head of molecular diagnostics at Holycross Cancer Center in Kielce, Poland. He envisions a time when every newborn is genetically screened for disease predisposition.

    According to Dr. Kowalik, enabling technologies that could make mass screening cheaper and faster are already in development—which is not to stay that valuable screening applications don’t already exist. Even currently available benchtop sequencers can make a difference in patients’ lives.

    The Holycross Cancer Center has a midsized laboratory that lacks the computational power of large genome centers. “Data analysis and clinical interpretation were our major roadblocks for NGS implementation,” continues Dr. Kowalik. “We had to choose a platform that generated just enough actionable data using commercial software.” A single benchtop sequencer, a panel of 50 genes, and a standardized validated workflow enabled rapid implementation of the NGS technology.

    Holycross uses NGS extensively to stratify patients for targeted therapy. Thyroid cancer is a common endocrine malignancy frequently associated with the oncogenic mutation in the BRAF gene (V600E).

    “When a patient presents with a suspicious diagnosis for malignancy from fine needle aspiration biopsy of thyroid gland, our endocrinologists routinely call for evaluation of the BRAF (V600E) gene by Sanger sequencing/qPCR,” remarks Dr. Kowalik. In one case, Dr. Kowalik recalls, Sanger sequencing did not reveal mutations in the BRAF. Instead, the Holycross NGS panel was used to uncover oncogenic mutations in a different gene (NRAS). This gene has a positive predictive value for malignancy of about 80%.

    Dr. Kowalik gave other examples when NGS assisted in finding mutations that may be overlooked by “gold standard” technologies. In Poland, 70–90% of inherited familial breast cancer results from only three founder mutations in BRCA1/2 genes. Prior to NGS introduction, the diagnostics was based on screening for these mutations only. Now Holycross has an ability to test the whole exome of BRCA1/2 genes.

    While the significance of all detected variants may not be yet known, they may still guide the placement of tested individuals into preventive programs. As the analytical capabilities of NGS grow and cost goes down, other family members may be tested for cancer predisposition. “Diagnoses made on the basis of whole-genome sequencing will relieve the uncertainty about breast cancer risk,” concludes Dr. Kowalik.


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