Updated on 17 March 2015
Dr Syamala Ariyanchira
Singapore: The next-generation sequencing (NGS) assays are expected to change the face of molecular pathology labs sooner or later. As key players announce their milestone achievements, the promise of NGS in this segment looks real and close. The technological advancements of NGS are also causing huge dilemma for the labs on various matters ranging from clinical interpretation of data to insurance coverage for the tests they would invest in.
Good news for NGS enthusiasts is that NGS is nearly attaining the Holy Grail of genomics-$1000 per genome! Platform companies racing to tap the clinical sequencing opportunities are driving innovation and bringing the platform closer to clinics. However, what percentage of the NGS hype will finally translate into market reality? And when it happens, how will it impact other molecular diagnostics segments that are currently dominant?
The Rising Business Focus on NGS Platform Development
Leading molecular diagnostic companies have already made it clear where their interests are, by channelizing their focus and investments into NGS to the exclusion of dominant fields such as PCR. Illumina was the pioneering company to initiate this business strategy, by announcing its exit from PCR in October 2013. Considering that the company entered PCR segment only in 2010 by acquiring Helixis, this move was perplexing at that time. However, the intention of the company became obvious soon after when MiSeq system of Illumina became the first NGS system to receive USFDA approval as Class II clinical diagnostics device. Further to this, Illumina also received clearance for three products for clinical use on MiSeq by USFDA. These milestone achievements in the NGS field reinforced the importance of Illumina's business strategy in addition to generating interest of the implications of the strategy in the molecular pathology field as a whole.
Other leading PCR companies are following suit and making strategic investments, though not completely abandoning PCR sector. Thermo Fisher Scientific recently completed listing its Ion PGM Dx NGS system with the FDA as a class II medical device for clinical use. Roche, on the other hand, closed down its 454 sequencing business and is exploring more promising NGS platforms to invest in. Bio-Rad recently acquired GnuBio for its droplet-based sequencing system while Qiagen is set to launch a benchtop sequencing system by 2015 based on the sequencing platform of Intelligent BioSystems, which it acquired in 2012.
As existing companies rewrite their strategies and new NGS-based start up companies join the clinical sequencing gold rush, a realistic assessment of the roles these tools in the molecular pathology space is essential to distinguish hype from realistic potential.
The NGS Milestones
The costs of NGS platforms are falling fast due to the intense competition in the field. In addition, the key players are developing innovative pricing strategies and range of products for catering to various categories of customers. For instance, Illumina's new HiSeq X Ten system, which is priced at $10 million, targets large volume users. The NextSeq 500 benchtop system priced at $250,000, on the other hand, targets customers with lower scale requirements. The MiSeq system is affordable by small budget labs at $125,000.
Several new single product companies are also contributing extensively in the NGS field by introducing innovative and affordable instruments. Oxford Nanopore Technologies, for example, is launching MinION, a nanopore-based sequencing device of the size of a USB memory stick, priced at $900.
Another milestone is the increasing read lengths achievable by NGS platforms. The short read length platforms employed for SNP detection would be useless for detecting gene variations that include large sequence deletions or insertions. Such regions may fall out during sample preparation and may not reach sequencers. While Illumina or Ion Torrent platforms offer up to a few hundred base pair read lengths, an average read length of 8,500 bases is achieved by Pacific Biosciences. Even MinION, one of the cheapest NGS devices announced so far, has an average read length of 5,000 base pairs according to Oxford Nanopore Technologies.
Technological advancements may not always translate into market share gains, though. Adoption by clinical labs will depend upon the real value addition by the platform against the proven tools currently in use. Hence, disease areas with unmet needs of great concern will be the pioneer fields where NGS assays will be adopted by molecular pathology labs.
Potential NGS Impact on PCR Demand
The PCR technology revolutionized molecular diagnostics since its entry in 1980s offering clinical labs a new possibility to make sufficient copies of target genes. Genomic DNA-based routine methods were soon replaced by PCR-amplified DNA. The emergence of high-throughput PCR systems enabled large scale analysis of patient samples. In one of our recent reports , we had estimated that the medical diagnostics accounted for about 43 percent of the global PCR technologies demand and forecasted it to grow at a CAGR of 8 percent.
PCR assays have limitations though, which may drive the adoption of more promising platforms such as NGS. A typical example is the inadequacy of PCR assays as standalone tools for diagnosing genetic diseases. PCR can lead to incorrect diagnosis if SNPs are present in the primer binding regions as it can cause allele dropouts during PCR reactions, which may go unnoticed. Sanger sequencing is often used as follow up test for confirmation, particularly if rare genetic disorder is a possibility.
Similarly, if deletions exist in the amplification region, only one chromosome will get amplified during PCR reaction. As a result, a heterozygous disorder may get diagnosed as homozygous. Additional methods to determine the carrier status of parents are required before arriving at clinical conclusions.
Infectious diseases sector is another area where PCR assays can fall short of meeting clinical goals. The diagnosis of multidrug resistant TB (MDR-TB) is a typical example. Though antibiotic susceptibility detection through PCR is achievable theoretically, few PCR assays are available in the market for routine clinical use. The real time PCR-based Cepheid Xpert MTB/RIF assay for rifampicin susceptibility detection is one of the few available at present.
Moreover, a single patient can be infected with multiple strains with varying antibiotic susceptibility. If PCR is used for identifying susceptibility to each antibiotic, multiple tests will have to be conducted. This is an expensive option and can be even fatal. Ideally, TB patients can benefit from identification of resistances to all antibiotics at once by accelerating the diagnosis and helping to start early treatment.
Another issue with PCR assays is the need to identify and validate critical biomarkers of a disease, which makes PCR assay designing and optimising a highly involved task. In clinical settings, this translates into the need to run several PCR reactions routinely to test each biomarker. This is not a viable option for a time-pressed clinical lab. Moreover, as efficiency of a PCR reaction decreases with the number of repeats, assay optimization for acceptable results is a challenge for the developers of PCR assays. False positives are additional concerns. Though TaqMan probe-based assays are more specific, creating individual probes for every allele of interest becomes an expensive task.
Clinical Microbiology and the NGS Advantage
NGS has a technological edge over PCR in clinical microbiology. In fact, this could be one of the fields to adapt NGS sooner since pathogen sequencing platforms have almost achieved the speed and the cost targets. Automated platforms that can interpret newly sequenced pathogen genomes in clinically useful manner are also emerging. Affordable NGS tools are also expected to lower reagent costs and save instrument time for labs. The potential to identify all the gene variants in a single experiment makes NGS faster than PCR-based genotyping. With automation, NGS tools can eventually be less laborious than Sanger sequencing - the current gold standard.
However, NGS replacing all the genotypic and phenotypic methods currently used by molecular pathology labs is not a realistic expectation as many of the current tools are competent enough clinically. However, some areas such as HIV and TB can benefit significantly from NGS when ready for routine clinical use.
Conventional phenotypic tests delay conclusive TB diagnosis due to the slow growing nature of the mycobacterium tuberculosis complex (MTBC). Though PCR assays assist to speed up TB diagnosis and even differentiate MTB from non-tuberculosis mycobacteria (NTB), antibiotic resistance determination remains to be a challenge. This critical gap can be filled by NGS assays that enable detection of resistance to all antibiotics simultaneously.
HIV is another disease where current diagnosis gaps can be fatal. For example, frequent monitoring of viral mutation is necessary to diagnose resistance emergence during HIV treatment. NGS-enabled viral tropism studies can provide clues on the mechanism used by HIV virus to enter host cells. Based on this, physicians can decide suitable antiviral therapy.
Clinical Utility Proof is Critical for NGS Adoption
Technological advancements can take a platform only to a certain extent in terms of market success. In healthcare, adoption largely depends upon affordability. In emerging markets where out-of-pocket payment is still the norm, even PCR assays are yet to be adopted widely and NGS adoption is not expected any time soon. In developed markets, clinical integration of NGS will be decided by the coverage and reimbursement policies of payers. Hence, even if a platform bridges critical diagnostic gaps, meeting the requirements of payers to prove clinical utility of the test is crucial.
The requirements by regulatory agencies to approve a test and launch it in the market is significantly lower than the payer requirements to prove clinical utility. Understanding and integrating these requirements early on during development phases will be essential for successful adoption NGS assays by molecular pathology labs. However, these requirements are unclear in this new emerging field. For the few available tests, clinical utility criteria vary widely among payers.
To complicate payment coverage issues further, NGS poses additional expenses related to incidental findings of genome sequencing and confirmatory tests that could follow these findings. Considering these regulatory and payment challenges, large scale switching by molecular pathology labs to NGS is not expected any time soon even if the field meets major technological challenges.