Need for speed in solid tumor molecular testing

The key to multitarget testing with rapid assays is in the multiplex capabilities, Dr. Arcila said, and multiplexing with qPCR is extremely difficult. SpeeDx (New South Wales, Australia) is one company with a solution, she said. “Utilizing two proprietary technologies, PlexZyme and PlexPrime, they can enable very high and reliable qPCR compared to other methods. In their primer-based amplification, they insert mismatches to the target parent sequence to make the PCR products for each target sufficiently different so you have more specificity for detection.” They combine this with the MNA enzymes, which are double enzymes that can use universal reporter probes. By combining the different probes and mismatches, “you can multiplex numerous targets in similar regions.” Some Idylla cartridges, for instance, are based on this technology, enabling detection of 51 and 21 different mutations in EGFR and KRAS, respectively.

More recently, a more flexible cartridge design began to be offered for the Idylla platform: a generic cartridge with liquid reagents for sample preparation, and dried reagents “where you can use vials with different designs with similar results,” she said. Her laboratory has been using it for the Idylla IDH1-2 mutation assay, with rapid results.

Dr. Arcila shared the case of a 65-year-old male who never smoked and had a 5.8-cm left upper lobe mass with extensive intrathoracic and cervical lymphadenopathy. By using pelleted cells from a pericardial fluid on one slide, they were able to determine this patient had a ROS1 fusion by expression imbalance and fusion-specific primers.

Dr. Arcila and colleagues also reported their experience with NSCLC cytology samples using Idylla’s ultra-rapid EGFR assay followed by NGS, primarily with MSK-IMPACT (Arcila ME, et al. JTO Clin Res Rep. 2020;1[3]:100077). They found that testing with the Idylla platform “enables rapid and accurate determination of the EGFR status without compromising subsequent NGS testing.” Prioritizing the assessment of EGFR mutation status by a rapid assay, the authors say, “would represent a critical step in guiding initial treatment decisions.” In general, nine percent of the cases tested by NGS had EGFR mutations not covered by the Idylla assay.

They evaluated the multitest approach in a second study of 1,240 biopsy samples and using the same Idylla EGFR assay as a screening method before NGS (Momeni-Boroujeni A, et al. J Mol Diagn. 2021;23[3]:310–322). Here, too, they found that use of the Idylla platform streamlined the assessment of the most common mutations in NSCLC while still allowing for comprehensive NGS.

With very targeted primer-based assays, Dr. Arcila said, the issue is covering all relevant insertions and deletions in lung cancer because they can be highly variable and difficult to multiplex. “As a rapid assay, we use fragment analysis to be able to rapidly screen these and make sure we’re not missing some of the rare alterations.”

For a rapid alternative to the Idylla platform, ChromaCode (Carlsbad, Calif.) offers high-definition PCR to highly multiplex qPCR and digital PCR applications. The HDPCR NSCLC panel is available off the shelf and includes the major targets for NSCLC with a minimum DNA input of 15 nanograms across two wells (about 7.5 ng per well). There is a third well for RNA targeting fusions, using five nanograms.

Similar to Idylla, the ChromaCode assay is designed to cover the most common indels, but does cover both ERBB2 and EGFR exon 20, “so you have to complement with additional non-NGS assays for more comprehensive assessment or wait for your next-generation sequencing platform results,” Dr. Arcila said. DNA and RNA extraction are needed for this platform; it takes about six hours (but depends on the method), and the extracted sample-to-result workflow can be completed in four hours. “You can have results for numerous key targets in the same day,” Dr. Arcila said, noting that this would not be possible if multiple serial qPCR had to be done. “You would rapidly deplete the tissue and it would take several days to complete.”

For testing by next-generation sequencing, Pillar Biosciences (Natick, Mass.) has a rapid platform with several NGS testing solutions, one of which is its pan-cancer OncoReveal solid tumor panel.

Pillar has more than 20 NGS testing kits in IVD or RUO formats, designed to scale on low-to-mid throughput NGS systems, Dr. Arcila said. Pillar recently partnered with Illumina to enhance its testing solutions, but the assays are agnostic and can be used with other sequencing platforms.

“The key to this technology is using the proprietary VersaTile, SLIM­amp, and PiVAT technologies,” Dr. Arcila said. The VersaTile primer design is AI-enabled, automated, and ultra-high plex. Pillar’s SLIM­amp enrichment chemistry involves a one-tube library preparation that eliminates the need for tube changes. “They have a standardized, single-day workflow, low-cost sequencing reagents, a very low assay failure rate, and it’s fully automatable and works well on damaged FFPE samples,” she said. Pillar’s PiVAT data analysis platform is the bioinformatics solution that incorporates what she describes as a highly effective error reduction algorithm to reduce sequencing errors, mapping errors, and other artifacts.

What sets this platform apart from others, she said, is that the technology inhibits the amplification of unwanted regions of the genome. To amplify large contiguous regions of the genome with PCR, primer targets have to be overlapped, “so you do not end up with uncovered areas. Usually those overlap areas are the ones that get the highest amplification and coverage because of the design.” Pillar’s stem loop structures inhibit the amplification of these areas, “so that you’re preferentially amplifying the regions you really want, so it gives you a very even coverage.”

Pillar is a two-day approach, Dr. Arcila said. Library preparation takes place during the first half of day one, followed by sequencing between days one and two, and analysis on day two. “Your analysis is about two hours.” The whole process can be completed within two to three days after extraction with this assay. With no transfers, “everything happens in a single tube, so you reduce the number of steps, which is key to decreasing errors, while also reducing time and associated labor costs compared with other conventional methods.”

The Genexus platform (Thermo Fisher, Waltham, Mass.) is a rapid NGS technology that provides rapid results with less complex workflows. With the Ion Torrent Genexus, Thermo Fisher has addressed major bottlenecks in comprehensive testing, Dr. Arcila tells CAP TODAY. It’s a two-instrument NGS system platform that automates and integrates the main steps of highly complex NGS workflows (sample purification, quantification, library preparation, sequencing, bioinformatics analysis and reporting). This facilitates NGS implementation in small laboratories, limiting the opportunities for human error and reducing variability of results.

Kojo S. J. Elenitoba-Johnson, MD, of MSK presented in the AMP session on the use of this system for hematologic malignancies and reported last year on the use of the Oncomine Myeloid Assay GX panel (Sande CM, et al. J Mol Diagn. 2023;25[2]87–93). In the context of lung cancer, the Oncomine Precision assay, which contains 50 genes, is designed to detect the most relevant mutations and fusions, with results available in two to three days.

Oxford Nanopore’s sequencing technology is the only one that offers a real-time analysis, Dr. Arcila said.

The formats are fully scalable, from pocket-size devices to population scale. “You can analyze native DNA or RNA and sequence any length of fragment and analyze not only the short cell-free DNA but also the ultra-long reads required for certain applications.” Sequencing from FFPE is not yet optimized but in the works for potential future use.

“This is an end-to-end platform that allows a lot of flexibility and control at every stage of the sequencing,” she said.

It is a PCR-free application “where you’re taking the native DNA or RNA and threading it through a nanopore. This incorporates a motor enzyme that threads that DNA or RNA, and you’re looking at about 400 base pairs per second that go through that pore,” she explained. “You’re capturing the electrical signal of the individual molecule as they go through, and it is the electro current variations that are ultimately translated into base calls.”

Oxford Nanopore’s sequencing devices range from the single-sample-capacity and portable ones such as the Flongle and the MinION, to high-throughput benchtop sequencers including the GridION and PromethION. “You can choose between simplex or duplex base calling models,” with the latter providing the highest quality and accuracy. “Simplex models allow greater output while duplex reduces the output and has more computational requirements,” Dr. Arcila said. Results are available within two to three days.

With molecular markers driving much of patient care, she said, “there is an equal need for rapid and reliable methods of analysis.”

And with the emphasis also on comprehensive assessment, “we often need to re-evaluate and change our approach to testing to be able to deliver results in clinically actionable times.”

As the opportunities for rapid testing through non-NGS and NGS assays increase, Dr. Arcila said, the creation of technology that’s easy to implement in local laboratories, with less dependency on extensive infrastructure, “has to be our push.”

Amy Carpenter is CAP TODAY senior editor.