This article has been updated to include additional information about the Qiagen QIAcuity system.
NEW YORK – Roche may be a bit of a latecomer to the digital PCR space with the recent launch of its Digital LightCycler system, but the company is banking on features such as high sample input volume, six-color detection, and easier custom assay development to help distinguish it in an increasingly populated market.
In particular, the company is targeting the platform toward the clinical oncology space, where its attributes may enable highly sensitive and multiplexed mutation-detection assays to aid in pharmaceutical research and, potentially, patient therapy guidance.
Roche first described the dPCR entry in 2018, and earlier this year, the firm described its expectation that the Digital LightCycler would be a competitive player in a market now including at least four other developers.
Launched last month for research use, the system boasts six optical channels and three different formats for plate-based digital PCR, including one called the HiSensitivity nanowell plate that accepts 45 microliters of sample.
"We continue to push PCR into more applications," said Miriam Fend, Roche's life cycle leader of PCR systems, noting that digital PCR will now allow the firm's customers to perform precision PCR testing, with "absolute quantification and unique sensitivity."
The Digital LightCycler system was developed by the Roche R&D team, Fend said, and the firm set out to make it different from other commercial offerings.
Currently, commercially available systems include the Bio-Rad Laboratories QX Droplet Digital line of instruments, the Stilla Technologies Naica system, Thermo Fisher Scientific QuantStudio Absolute Q Digital PCR System, and the Qiagen QIAcuity. These differ in aspects such as the way droplets are formed for PCR, the number of instruments required, the number of optical channels available, and the overall workflow and time to results.
A few of the competing systems use emulsion-based droplet formation, but the Roche system is plate-based. Fend said this makes it simpler to use and broadens access for labs that need more standardization and an easier workflow, like clinical labs.
The system features an automated workflow that divides sample into 100,000 partitions, and can produce results in 1.5 to 3 hours, Fend said. It also features what Fend called "intuitive" software for data analysis and results reporting, and can be connected to LIS systems.
The form factor is also a critical part of the Roche brand, according to Fend, and the Digital LightCycler looks and feels like other Roche instruments. And, it completes the firm's offering of molecular systems, covering the qPCR spectrum from the point-of-care Liat to the high-throughput Cobas 6800/8800.
Roche can now offer customers "a full suite of PCR technologies" that has a "look, feel, and user experience" that makes it more seamless to switch between technologies, Fend said.
Clinical oncology push
Digital PCR has the benefit of providing quantification without requiring separate calibration steps, which helps to conserve precious samples. Multiplexing in multi-channel systems also amplifies this attribute.
One place that high accuracy and multiplexing could be useful is in testing for genetic mutations in heterogeneous tumor samples for clinical oncology. Fend said the Digital LightCycler offers three different plate consumables, with a novel deep well plate that can potentially enable detection of low-concentration or rare targets using fewer reactions.
In April, Roche presented a poster at the American Association for Cancer Research annual meeting describing use of the Digital LightCycler's six-channel capabilities and deep well plates to enable a 43-target, two-tube assay for simultaneous detection of EGFR mutations.
The team noted that the test detects whether T790M and C797S mutations are in cis or trans phase in the same reaction, and also reports the allelic context by detecting and quantifying other EGFR mutations, including 27 deletions in exon 19 and six insertions in exon 20.
Such a test could have applications for determining whether a patient with non-small cell lung cancer has mutations necessitating a switch to third-generation tyrosine kinase inhibitors, according to the poster.
Specifically, half of patients on first- and second-generation TKIs develop a T790M resistance mutation, and can then be switched to the third-generation TKI called osimertinib (AstraZeneca’s Tagrisso). However, this subsequent treatment can lead to the emergence of a C797 mutation in the EGFR gene. If this mutation is in a trans phase, combining first- and third-generation TKIs can have some benefits for these patients.
Whether the overall allelic context matters in this treatment is unknown, however, and Roche researchers said in the poster that a multiplex EGFR mutation panel could thus be a useful research tool.
The firm reported that the EGFR multiplex assay could detect mutations at frequencies as low as 0.1 percent in contrived cell-free DNA samples.
Roche also makes a qPCR test, the Cobas EGFR Mutation Test v2, which detects a total of 42 targets and was used as the comparator in the Stilla study. That test has been cleared by the US Food and Drug Administration for use as a companion diagnostic for five different TKI therapies.
In the AACR poster, Roche researchers compared their EGFR panel to an assay from Bio-Rad called the Bio-Rad Expert Design assay, which runs on the QX200. They found that both assays had similar performance detecting cis versus trans mutations, but noted that the Bio-Rad test only detects C797S and T790M.
Of note, Bio-Rad's QX200 system has two optical channels, while Thermo Fisher's Applied Biosystems QuantStudio Absolute Q Digital PCR System has four optical channels with a fifth used for reference. Bio-Rad is also currently developing a six-channel, 100,000 droplet system called the QX600.
The Qiagen QIAcuity system, meanwhile, is available as one-plate instrument with either with two or five channels, and the firm also makes four- and eight-plate instruments in which five channels are standard, according to a spokesperson.
Fend said that Roche has also formed a collaboration with MilliporeSigma to develop custom oligonucleotides for customers developing their own tests. And, although the firm is oriented toward clinical applications at the moment, she noted that the system could also be used by customers for other applications such as wastewater testing or biopharmaceutical quality control for things like gene and cell therapy manufacturing.
However, "where we really think that digital PCR has its largest potential is moving into a clinical space to provide oncology and disease monitoring testing, and that is where we at Roche are really strong," Fend said.
Mikael Kubista, founder and CEO of TATAA Biocenter, is among the early users of the Digital LightCycler, and has been testing out the system for about four months, he said in an email.
"We have all the leading dPCR platforms providing hands-on training courses to the research community regularly," Kubista said.
The team also coauthored the dPCR MIQE guidelines to teach researchers how to plan, perform, and report dPCR studies, and it contributed to the recently released ISO standard on qPCR and dPCR, he also noted.
Because TATAA Biocenter is an accredited laboratory, it must perform extensive testing of new instruments, validating performance and establishing workflows. TATAA also integrates them into a LIS system it uses, called Benchling.
The primary applications for dPCR in the TATAA lab are pharmacodynamic and pharmacokinetic studies, rare sequence detection, and calibration of standards.
"We are also establishing standard operating procedures for the design and validation of custom dPCR assays," Kubista said.
In this setting, a "unique and highly appreciated feature" of the new Roche dPCR system for Kubista is its separated workflows for development and regulated bioanalyses, he said.
"The development mode offers the flexibility we need when designing and validating custom assays for clients' PK/PD studies, while the regulated bioanalysis mode satisfies the requirements for GLP studies and enables efficient technology and method transfer when requested," he said.
As someone familiar with other commercially available digital PCR systems, Kubista said they are "all good," but there are some distinguishing features of the Roche system.
In particular, the three different plates allow for cost and performance optimization, he said.
"The high sensitivity plate accepts an impressive total volume of about 45 microliters," he noted, which he said is important because the sensitivity to detect a rare event is proportional to the total sample volume analyzed.
In addition, "the high-resolution plate has an impressive 100,000 partitions," he said, adding that "corresponding analyses on other instruments typically require multiple runs at higher cost."
Finally, the easy integration into the LIS system is also "a critical advantage" for laboratories performing accredited analyses and working according to GLP or GCLP, he said.
Kubista said that it is "straightforward" to design custom assays using the Digital LightCycler, "including assays based on advanced designs, such as two-tailed PCR that enhances specificity and allows for the detection of much shorter targets."
For carrier genetic testing services and many diagnostics applications, this attribute is critical as customers often require customized assays, Kubista said.
"Some dPCR instruments require proprietary complex master mixes, which complicates custom design for service providers," he also said.
Regulated content and standardization will now be a critical differentiating factor among dPCR instruments, Kubista said, adding that it will also be important to offer customized solutions based on validated assays for certain diagnostic applications.
"We expect [dPCR] will revolutionize several areas in diagnostics," he said, anticipating breakthroughs in cell-free DNA analysis for cancer, graft rejection, and noninvasive prenatal testing, as well as forensics and archaeo-genetics. And, digital PCR is poised to lead to biology research breakthroughs, and to spur the development of the pharmacodynamic and pharmacokinetic assays needed to advance cell and gene therapeutics, Kubista noted.