Next-generation sequencing (NGS) applied to genomic testing has provided clinicians with a powerful tool to better diagnose patient samples. NGS offers the advantages of high throughput and smaller sample requirements compared with its predecessor, Sanger sequencing. The past few years have seen a rapid expansion of genomic testing capabilities at medical centers using NGS for whole-genome sequencing (WGS), whole-exome sequencing (WES) and targeted sequencing applications. Today, researchers and clinicians can use NGS to test several hundred genes for mutations, copy number changes and fusions. Here’s a look at how NGS is used in the clinic, and how companies are offering NGS tools to meet both clinical-research and diagnostic needs.
Using NGS for clinical decision making
Specific genomic alterations that affect the course of treatment—or actionable alterations—are the most important for clinicians. Many clinicians use genomic testing only when it’s useful in guiding treatment. Although only a handful of genetic alterations are linked to an approved drug, another option is clinical trials. “We have almost 100 clinical trials that require that the patient’s tumors have a specific genetic alteration,” says Funda Meric-Bernstam, chair of investigational cancer therapeutics at the University of Texas MD Anderson Cancer Center.
Most clinical decision making today is guided using exome-based NGS panels that look for specific alterations related to a patient’s condition. Some NGS panels test for a collection of mutations, such as panels for breast cancer or solid tumors. Small gene panels, which are often used to test for specific phenotypes or classes of tumors, typically yield clear results of limited complexity. “This makes them usable by most all physicians with the appropriate academic domain expertise,” says Matthew Ferber, a clinical molecular geneticist and lead director for the Clinical Genome Sequencing Laboratory at the Mayo Clinic. In contrast, large gene panels and WES require a larger interdisciplinary team to manage the process, from initially meeting with the patient to interpreting the final results. Most clinicians don’t routinely use whole-genome sequencing—which takes longer, costs more and creates bioinformatics and data storage issues—because it usually doesn’t yield any further actionable information to benefit the patient (see below for exceptions).
When it comes to diagnosing patients, the consequences of errors can be drastic. So clinical labs spend a lot of time confirming changes on alternate platforms, using either a different NGS system or the established Sanger method. “Still, the gold standard is Sanger sequencing,” says Jaclyn Biegel, director of the Center for Personalized Medicine and division chief of genomic medicine at Children’s Hospital Los Angeles and a professor of clinical pathology at the University of Southern California, Keck School of Medicine. “For whole-exome sequencing in a clinical diagnostic setting, where you’re trying to make a diagnosis for a child with a rare disorder, we confirm pathogenic variants by Sanger.”
Ulrike Kappes, director of the Developmental and Neurogenetics Laboratory in the Human and Molecular Genetics Center at the Medical College of Wisconsin, also uses Sanger sequencing to confirm NGS results. Kappes’ team uses NGS in whole-exome and genome sequencing to diagnose rare diseases, such as neurodevelopmental disorders. “Exome and genome sequencing are part of the clinical work-up for our patients when a genetic component is strongly suspected, but other tests have failed to provide a diagnosis,” says Kappes.
Biegel’s group is developing the use of whole-genome sequencing for pediatric oncology specimens, to test for chromosomal rearrangements—something that exome sequencing often fails to detect. “We’d miss too many things using just whole-exome sequencing, so for somatic detection of rearrangements in cancer samples, we’re just going to leapfrog right to whole-genome sequencing,” says Biegel. They use a panel first, though, to avoid the complex bioinformatics that would be required to perform whole-genome sequencing on every patient.
Expanding the reach of NGS
Much of the current NGS testing is tissue-based, but greater interest in liquid biopsies is driving innovation in this technology.
Advantages include noninvasive sampling (such as a blood draw) and the possibility of serial sampling to study a patient over time. Blood samples are much easier to procure than biopsies of inaccessible tumors. For patients previously treated elsewhere, transferring an archival tissue sample can delay therapy, and performing a new biopsy is more invasive and costly. “Also, about 15% of patients who undergo biopsies don’t have enough tissue to facilitate genomic testing,” says Meric-Bernstam. “Having the option of liquid biopsies would increase the genomic testing options for patients.”
Cynvenio has developed a means of evaluating tumor signals from liquid biopsies that uses tumor-derived cells and DNA fragments present in a patient’s blood. Cynvenio’s platform enriches tumor cells and purifies two types of DNA from blood—circulating cell-free DNA (ccfDNA, DNA released from dead or dying cells) and circulating tumor cell DNA (ctcDNA, DNA from intact cells that originate in the tumor and enter the blood)—which provide different kinds of information. Cynvenio also developed a triple-negative, breast-cancer-specific sequencing panel that lets researchers look for hallmarks of this disease in blood samples by sequencing both ccfDNA and ctcDNA.
Currently Cynvenio is using these tools in a study of patients in remission from triple-negative breast cancer, an aggressive disease with 30% recurrence. The company uses NGS evaluation of blood samples over time to monitor for molecular evidence of breast cancer recurrence. The clinical trial seeks to establish links to clinical recurrence. CTCs can be detected early, even before clinical manifestations. “That information can be used to provide evidence-based information that can help the doctor make a decision about … the best way to move forward for each patient,” says Paul Dempsey, chief scientific officer at Cynvenio.
NGS tools for future applications
Researchers and clinicians continue to push the boundaries of current NGS technology. For example, parts of the human genome remain difficult to sequence using NGS. “These include copy number variations, long repeat sequences, structural chromosomal rearrangements, polyploidy, GC-rich regions, epigenetic effects or mosaicism,” says Kappes. NGS is also limited when it comes to identifying variants in regions of the genome with high sequence homology to other regions, such as paralogous genes and pseudogenes. “For certain genes, and specifically certain exons of various genes, we still have insufficient coverage or read depth to analyze these regions,” says Kappes.
Researchers are working jointly with companies to develop innovative tools and assays that play to the strengths of NGS in the clinic.
Biegel is working with Thermo Fisher Scientific to develop an NGS panel for pediatric oncology patients. “Thermo Fisher’s AmpliSeq Oncomine research panel didn’t include sufficient content for childhood cancers,” says Biegel. Her group is co-developing a comprehensive NGS panel that includes biomarkers for all pediatric tumor types. Still being validated, the panel uses Thermo Fisher's high-throughput Ion Torrent NGS platform and AmpliSeq technology; it requires a starting sample of only nanograms. “It’s a combined DNA and RNA platform, so we can pick up the translocations as well as the mutations,” Biegel says.
Some NGS tools, though designated for research use only, are increasingly valuable in the development of clinical tools. Promega offers research-use-only tools to quantitate DNA going into a library and is developing a quality-control assay to ascertain the level of sample degradation. Knowing the quality and size of the sample DNA helps researchers get the most information possible from a sample. “When researchers work with poor quality and precious samples, for example, they might decide to use a smaller [NGS] panel of only the most important genes to get the most useful information they can,” says Curtis Knox, global strategic marketing manager for NGS at Promega. Amid greater interest in liquid biopsies, Promega also recently released a method for purifying circulating cell-free DNA that uses the Maxwell® RSC ccfDNA Plasma Kit on the Maxwell® RSC Instrument.
QIAGEN’s GeneReader system, currently for research use only, helps clinical researchers narrow their search for actionable information. “The purpose of a research-use-only [instrument] is to enable the development of future diagnostics tools,” says Jonathan Arnold, oncology franchise business leader at QIAGEN. “We want to drive NGS into the clinical routine.” QIAGEN’s QIAact Actionable Insights Tumor Panel targets 12 genes and up to 1,250 genetic mutations in common cancer types. Recently the company released the panel for use with liquid-biopsy plasma samples. QIAGEN also is developing additional QIAact panels for lung cancer and for breast and ovarian cancer.
Although sequencing costs continue to fall, cost is still a limitation impacting wider adoption of NGS in clinical situations.
A more complex hurdle is the need for complicated systems and interdisciplinary teams of experts to interpret the NGS results in a clinical setting.
"[NGS testing] won’t take off in a major way until someone invents an even cheaper and faster sequencer that, more importantly, can be managed by an individual or two from sample prep to variant interpretation and through report generation,” says Ferber. “Someday, I would hope that the data analytics requirements would become far less cumbersome to manage.”
Ferber’s program at the Mayo Clinic has grown quickly to accommodate a growing volume of requests for NGS testing. “It’s expensive, hard and time-consuming, but all that fades away when working through a case that hasn’t been solvable for over 10 years—forcing families into countless doctor visits and spending untold amounts of money—and [then] the team finds the answer,” he says. “That’s when you know it’s all been worth it.”