Clinical NGS Finding Its Groove

Clinical NGS is the use of genomic information generated by next-generation sequencing methods to diagnose or stage disease or to inform the selection of therapy and monitor its effects.

Though far from routine, NGS is increasingly being integrated into medical practice, and makers of products that address the NGS pipeline are actively marketing to clinicians while carefully working through an unsettled regulatory environment and advocating for reimbursement of NGS-based laboratory tests.

Applications of clinical NGS include the molecular characterization of tumors, germline analysis for disease susceptibility, and identification of disease-causing organisms. Comprehensive genomic profiling of tumors using NGS can detect mutations predictive of chemotherapy response and prognosis.¹ Germline analysis by NGS can be predictive of disease susceptibility, enabling pro-active management of clinical risk.² In cases of infectious disease, NGS enables the detection and identification of pathogens not identified by routine diagnostic assays, directly from clinical samples.³

Making room in the lab

Clinical NGS does not necessarily replace previously adopted genome analysis methods such as quantitative PCR and microarray analysis. Rather, it is finding a place alongside those methods. Quantitative PCR is the gold standard of quantitative accuracy, but both microarray analysis and NGS offer much higher throughput, enabling the testing of hundreds or thousands of loci in a single reaction, versus the single-locus limitation of qPCR.¹

The principal clinical advantage of NGS over both microarray analysis and qPCR is the ability of NGS-based diagnostic tests to assess large segments of the genome and to detect variants in an untargeted way.⁴ NGS and microarray analysis are fundamentally dissimilar in that microarray data are produced by complementary DNA strand recognition, whereas NGS data are produced by DNA strand synthesis. In microarray analysis, DNA loci of potential clinical importance must be predicted in advance and be present in the form of an opposite-strand probe on the microarray surface. By contrast, in NGS, even if the sequence of a locus of interest varies from the expected sequence, it will still be synthesized accurately and detected during the variant analysis phase of the sequencing project.

The principal clinical advantage of NGS over both microarray analysis and qPCR is the ability of NGS-based diagnostic tests to assess large segments of the genome and to detect variants in an untargeted way.

This unique quality of NGS is of particular interest in oncology, where the ability of cancerous cell populations to mutate poses a major challenge. Clinical NGS analysis of tumors uncovers the small changes characteristic of these mutations—insertions, deletions, and point mutations—as well as larger changes such as rearrangements and copy number changes.

Another reason clinicians are making room for NGS is that there is no hybridization-based equivalent to whole-genome or whole-exome sequencing. The targets of an NGS-based test can range from mutational hotspots to the entire genome, enabling the correlation of disease states with previously unknown genomic variations, as well as the detection of rare variations for which no microarray probes exist.

FDA-approved tests

In the U.S., diagnostic tests provided to clinical laboratories are regulated by the U.S. FDA. However, only a handful of NGS-based diagnostic tests have been approved. Two tests have been approved this year: In June, Thermo Fisher Scientific obtained FDA approval for the Oncomine Dx Target Test, which can be used to identify best responders to AstraZeneca’s EGFR inhibitor Iressa (gefitinib), Pfizer’s ALK and ROS1 inhibitor Xalkori (crizotinib), and the combination of Novartis’ MED inhibitor Mekinist (trametinib) and RAF inhibitor Tafinlar (dabrafenib). Also in June, Illumina received FDA approval for the Extended RAS Panel, which helps clinicians identify which patients are eligible for treatment of metastatic colorectal cancer with Amgen’s Vectibix (panitumumab). In December 2016, Foundation Medicine obtained FDA approval for the company’s FoundationFocus CDxBRCA test, a companion diagnostic to aid in identifying women with ovarian cancer who may benefit from treatment with Clovis Oncology’s poly(ADP-ribose) polymerase (PAR) inhibitor Rubraca (rucaparib).

Previously, in 2013, Illumina obtained FDA clearance for two cystic fibrosis assays and a “universal” kit for developing new NGS-based molecular diagnostic tests.

These kits, which are eligible to be sold by their manufacturers for use in clinical laboratories throughout the United States, represent only a small fraction of the clinical NGS market. The vast remainder of clinical NGS takes place in CLIA-certified labs as laboratory-developed tests.

The long tail

A laboratory-developed test (LDT) is an in vitro diagnostic that is made and used only within the lab that developed it. When a clinician orders an LDT, the laboratory offering the test receives the patient specimen, runs the test, and reports the results back to the physician.

While the FDA regulates tests sold as kits, laboratory-developed tests are separately regulated by the Centers for Medicare and Medicaid Services (CMS) under the Clinical Laboratory Improvement Amendments (CLIA), which apply to approximately 250,000 patient sample-testing laboratories in the United States.⁵ Just how many LDTs are actually on the market is difficult to estimate, but the number is significant. Uniquely, in New York State, LDTs are reviewed by the New York State Department of Health through a permit application process. In 2014, 565 laboratories submitted 9,800 LDTs for review.⁶

Ambry Genetics, Caris Life Sciences, Foundation Medicine, GeneDx, and Invitae are some of the better-known organizations, among many others, offering NGS-based LDTs. Both Illumina and Thermo Fisher Scientific also operate CLIA-certified laboratories offering clinical NGS services.

New tests come on the market frequently. For example, in the past year, NGS-based LDTs for women’s reproductive health, inherited genetic disorders, and genetic alterations associated with cancer have been introduced by Celmatix, Good Start Genetics, and Personal Genome Diagnostics, respectively. Clinicians working in academic centers operating their own CLIA-certified clinical laboratories may have access to a specialized menu of LDTs developed in-house.

Getting started

The steps involved in performing an NGS-based clinical test fall into three categories: The pre-analytic phase includes sample collection, shipping, de-identification, and laboratory processing. The analytic phase is composed of DNA sequencing, alignment, and base calling. Variant calling, interpretation, report generation, delivery, and data storage comprise the post-analytic phase.

At present, the market for instrumentation used in the analytic phase is led by Illumina and Thermo Fisher Scientific. Buyers interested in outfitting a CLIA lab for clinical NGS may also consider systems from Pacific Biosciences and Oxford Nanopore for their unique advantages. Up-and-coming but unreleased NGS systems are on the way from GenapSys, Genia, and others.

Few would argue that the most important consideration when setting up a lab for clinical NGS is the human element—the cost and availability of specialist technical expertise required to prepare samples and run the instrumentation. If the intention is to supplant certain front-end manual protocols with automation, technical fluency with automation will also be required. In addition, the capabilities, throughput, and range of sample batch sizes produced by the automated sample processing system must match the capabilities of the paired sequencing systems as well as the anticipated laboratory workflow.

Few would argue that the most important consideration when setting up a lab for clinical NGS is the human element…

The technical capabilities of the lab must also correlate with the types of samples that will be run. With current technologies, liquid samples such as blood are considered simplest to prepare, while the preparation of samples such as FFPE solid tumors is more difficult to reduce to a protocol that will work with all incoming samples.

Running efficiently

Although NGS-based LDT marketing materials often specify turnaround times in weeks, faster return of results is already becoming a competitive differentiator for some tests. One can imagine that in the near future, STAT clinical NGS sequencing will become a reality, such as when an effective antibiotic must be quickly selected to treat a life-threatening infection of unknown etiology. In the case of standard orders, the ability to deliver results within a few hours, while the same day-shift medical staff is on-duty, will improve patient care.

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As clinical adoption of NGS proceeds, dissimilarities between the sequencing needs of clinical laboratories and those of the original NGS market of academic center core facilities will become increasingly apparent. One readily predictable difference is in the average number of samples per run. Unlike a core facility, which can optimize lab efficiency by allowing samples to accumulate for some time before starting a run, a clinical NGS lab may need to run a single sample on a STAT basis, potentially more than once a day. In that case, two or more stand-alone systems optimized for individual samples may suit a clinical laboratory’s workflow better than a larger system that can efficiently sequence dozens of samples simultaneously.

Trends

More panels for more indications

As our knowledge of the human genome increases, so do the viability of precision medicine and the clinical value of NGS. More gene panels covering more indications are certainly forthcoming. For example, BioMarx Therapeutics has announced its intention to introduce an NGS-based test for early diagnosis of pancreatic cancer. Pancreatic cancer is treatable, but is rarely diagnosed before cancer has already spread beyond the pancreas and prognosis is poor. If high-risk individuals can be identified and tested, this LDT will save lives.

Faster turnaround

As the number of CLIA labs offering similar diagnostic panels increases, they will seek ways to differentiate their services. For example, the StrandAdvantage test offered by Strand Life Sciences, delivers a preliminary report on mutations affecting genes related to therapeutic drug response in 10 days, allowing care providers to consider genomic information when selecting first-line treatment for cancer.

Greater use of whole-genome sequencing

Today, the use of a targeted gene panel suggests that some time has passed since initial presentation, and a relevant body of diagnostic detail has already been acquired. A gene panel test can then provide additional information to confirm, or contradict, a specific diagnostic conclusion. By comparison, whole-genome sequencing could save time, reduce the need for other lab tests, and bring to light valuable diagnostic information that falls outside of existing diagnostic decision matrices.

Earlier utilization

When clinical NGS tests are ordered, it is usually late in the diagnostic process, sometimes after initial lines of treatment have failed. This could be due, in part, to the low level of CMS reimbursement for sequencing tests versus their costs. This difference will narrow as the cost of clinical NGS descends to reimbursement levels. What may take longer is the process of establishing a place for clinical NGS in the medical training curriculum. Routine use of clinical NGS early in the diagnostic process is unlikely to happen before care providers become familiar with its value, know what tests are available and how to order them, and how to use the information they can provide. To this end, the National Health Service of England has launched the Genomics Education Programme to ensure that its staff has the knowledge, skills, and experience required to capitalize on NHS investments in Genomic Medicine Centres and the 100,000 Genomes Project.

We are in the early days of clinical NGS, but few would question its ultimate value. When the cost of patient-sample DNA sequencing is equal to the CMS reimbursement level, and the process of NGS can be carried out in a CLIA lab without hands-on intervention by specialized technical personnel, and physicians know what tests are available to them, when to use them, how to interpret the results and how to use those results to help their patients get better, faster, the promise of clinical genomics will be fulfilled.

References

1. Sen, M, et al., “StrandAdvantage test for early-line and advanced-stage treatment decisions in solid tumors,” Cancer Med-US, 6(5), 883-901, 2017. [PMID: 28371134]

2. Ballinger, M, et al., “Monogenic and polygenic determinants of sarcoma risk: an international genetic study,” Lancet Oncol, 17(9), 1261-1271, 2016. [PMID: 27498913]

3. Gong, Y, et al., “A metagenomics study for the identification of respiratory viruses in mixed clinical specimens: an application of the iterative mapping approach,” Arch Virol, 162(7), 2003-2012, 2017. [PMID: 28424887]

4. Petrone, J, “FDA wades into sequencing-based diagnostics regulation,” Nat Biotechnol, 34(7), 681-682, 2016. [PMID: 27404865]

5. “Clinical Laboratory Improvement Amendments (CLIA) - Centers for Medicare & Medicaid Services,” Cms.gov. 

6. “FDA’s LDT proposal means ‘whole new ballgame’ for labs,” CAP TODAY. 

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