NEW YORK – Stilla Technologies is progressing on a comprehensive strategy of standardizing and decentralizing digital PCR for clinical use. Having initially focused on the genomics research market, the firm has lately been adapting its software, hardware, and content with an eye toward serving clinical end users.
This week, Stilla announced the launch of 20 new oncology assays for its Naica digital PCR system developed in partnership with ApexBio. These new tests are in addition to 10 kits the firm already developed and come on the heels of an announcement last month that Stilla will partner with Atila Biosystems to codevelop as many as 20 liquid biopsy assays in 2023.
Among the recent adopters of Stilla's technology for oncology testing is Véronique Quillien, director of the biology department at the Eugène Marquis Cancer Center, a medical center in Rennes, France.
Quillien and her team recently developed a 31-plex liquid biopsy test on the six-color Stilla system enabling them to screen circulating tumor DNA of metastatic breast cancer patients for ESR1 and PIK3CA mutations.
Early detection of ESR1 and PIK3CA mutations is key to better personalizing the management of patients with hormone receptor positive, human epidermal growth factor receptor 2 negative (HR+/HER2-) metastatic breast cancer, Quillien said in an email.
The team had previously developed two single-well multiplexed assays that detected either 11 ESR1 mutations or 21 PIK3CA mutations, publishing the latter in Scientific Reports. But, the debut of the six-color Naica system in 2021 allowed the team to combine these two assays.
In a poster presented at the Molecular Analysis for Precision Oncology Congress in October, the team presented data on their use of this lab-developed test. They noted that ESR1 and PIK3CA analyses are often requested together, so this new screening assay represented significant time and resource saving for the lab and for metastatic breast cancer patients "with a relative ease of implementation and analysis."
In their sample, the team found that ESR1 mutations had a frequency of 28 percent, while PIK3CA mutations had a frequency of 32 percent. The panel results were generated in less than three hours in a decentralized hospital lab using 4 milliliters of blood.
The results of the test could allow oncologists to alter a patient's treatment, Quillien said, for example, by switching them to alpelisib, an isoform-specific PI3Kα inhibitor approved for PIK3CA-mutated patients with HR+/HER2- locally advanced or metastatic breast cancer patients.
The team's use of liquid biopsy also has advantages, and Quillien added that there are several arguments for assessing the PIK3CA status in metastatic cancer cases. These include the risk of genomic evolution between the initial and metastatic tumor, and the risk of poor quality or quantity of DNA extracted from archival biopsies of formalin-fixed paraffin-embedded primary cancers that have been stored for a long time.
"Since obtaining a metastatic tissue biopsy can be challenging for the clinician [and] uncomfortable for the patient, ctDNA assessment is an effective alternative to metastatic tumor analysis," she said.
ESR1 mutations are also an interesting emerging clinical biomarker for HR+ metastatic breast cancer, Quillien said.
"These mutations are usually acquired during endocrine therapy — particularly after treatment with an aromatase inhibitor — which leads to an enrichment at the metastatic stage of the disease compared to the initial stage," she said.
Recently, a randomized Phase III trial called PADA-1 has shown that monitoring ESR1 mutations in ctDNA can be useful to trigger the switch from aromatase inhibitor treatment to fulvestrant in HR+/HER2- metastatic breast cancer patients, Quillien said. "Since then, ESR1 testing has begun to be integrated into patient care in several oncology centers," she added.
The team initially chose the Stilla system because, when it began this work in 2019, Stilla's was the only dPCR instrument that allowed the use of three colors to enable higher multiplexing. The team has also used the three-color Naica system to develop assays to detect KRAS and NRAS mutations for colorectal cancers and BRAF mutations for melanoma and colorectal cancer.
Since 2019, the team has performed approximately 1,500 digital PCR assays for patient follow-up, Quillien said. After adopting the six-color system, they have also seen the benefits of higher multiplexing on top of the high sensitivity and robustness of digital PCR and the support of the Stilla technical team, she said.
A twist on dPCR
Some of the assays Quillien and her team developed have also incorporated a twist on standard digital PCR. This new design strategy, called drop-off, enables the simultaneous detection of multiple mutations within genomic hotspots, she said.
Specifically, a reference probe is designed to target an invariable region close to the mutational hotspot, and a drop-off probe is designed to target the wild-type sequence of the hotspot region.
"A double positive fluorescence signal indicates the presence of a wild-type allele," Quillien said. In the case of a mutant allele, the binding of the drop-off probe is lost, which results in a lower fluorescence amplitude, "with droplets clearly distinguishable from those containing wild-type alleles," she said.
This allows the simultaneous detection of several mutations including the 21 PIK3CA pathogenic mutations, 11 ESR1 mutations, and 17 ERBB2 alterations including insertions and point mutations.
"For these three assays, we have a multistep diagnostic strategy — we perform a screening assay first, [and] if there is no evidence of mutation, samples are considered negative," Quillien said. For a positive sample, targeted assays are performed in order to precisely identify the mutation or mutations present in the sample, she added.
Prior to developing the ESR1 and PIK3CA assays, the center did not screen for these mutations, Quillien said. Now, going forward with the Stilla system, the team is "currently developing an assay for colon cancer that will allow the simultaneous detection of KRAS/NRAS and BRAF mutations," she said.
New hardware, software, and content
The pivot to a more clinical orientation was initiated by CEO Philippe Mourere when he joined the company and includes the cell and gene therapy market as well as the clinical lab space.
"Over the past two years we have realigned our portfolio to better serve the downstream community," Mourere said in a recent interview.
For cell and gene therapy, Mourere said digital PCR lends itself to the stringent needs for quality control. Developers need to characterize their transgenes and verify that all cells were transfected correctly, for example. The US Food and Drug Administration has also been raising the bar on biopharma developers, he said, and these end users require impeccable quality, in part because their biological products cannot be sterilized after they are made.
"We really recognized a couple of years ago that these two communities have a common realm, in the sense that they have a clinical play," Mourere said. "In order to serve these two markets, we had to deal with sample traceability, sample [prep] automation, and compatibility with workflows."
These markets are also much more exposed to regulatory and workflow constraints, he said, and end users tend to have different skill sets than those in research labs. Stilla had to adapt its Naica digital PCR system to the needs of clinical reference and biopharma labs for rapid, end-to-end, push-button solutions, he added. So, in addition to growing its menu of assays to help standardize testing, the pivot also entailed adapting both the hardware and software of the Naica system.
First debuted in 2016, Stilla's digital PCR approach encourages droplets to self-arrange into a hexagonal mesh that resembles a crystal. The firm has since increased the number of samples that can be processed simultaneously in these crystals from four samples on its legacy Sapphire chip to 16 samples on its newly launched Ruby chip.
The firm also adapted the structure and spacing of the wells on its Ruby consumable so that it can be incorporated into automated liquid handling workflows. The Ruby is 96-well plate-compatible and is covered in aluminum foil that can be pierced by the end user, either manually or with a robotic liquid handling system and a multichannel pipetter. This foil reduces the possibility of cross-contamination of wells, Mourere said, adding that the chip is also barcoded to facilitate traceability.
In addition to these hardware changes, the team realized that the liquid biopsy space in particular is also becoming increasingly highly regulated, Mourere said.
Stilla therefore adapted its software to be compliant with part 11 of Title 21 of the Code of Federal Regulations; Electronic Records; Electronic Signatures, otherwise known as 21 CFR part 11. This new software allows the sample to be traced throughout the workflow, but is also user-friendly so it can integrate with the end users' LIM system. The team has now completed a number of 21 CFR site implementations, and "it's working very nicely," he added.
For the workflow piece, Stilla has partnered with Promega to allow the system to integrate into automated workflows. In a poster presented at the American Association for Cancer Research conference this week, the partners showed that there was a high compatibility between the automated Maxwell extraction system, the Maxprep Liquid Handler, and multiplex detection from multiple sample types on the six-color Naica system using the Ruby chip.
According to the poster, this complete workflow allowed for repeatable detection and quantification of human RNAs across a range of sample concentrations and sensitive detection of the main somatic EGFR mutations described in non-small cell lung cancer.
"The combination of these technologies has a fast time-to-result with minimum hands-on-time, enabling a complete sample-to-result workflow in less than a day," the poster authors wrote, noting that the proof-of-concept workflow creates a foundation for further development of streamlined sample-to-answer oncology protocols.
Stilla also plans to develop similar collaborations with other automated liquid handler suppliers, Mourere said.
In terms of additional menu for these workflows, Stilla intends to continuously release fully automatable kits in partnership with assay developers, Mourere said. The firm has developed a partnership program that allows these developers to add their content co-branded with Stilla, "so that their brand doesn't disappear," he said. The assays in turn have the advantage of standardizing testing so that it can be performed at many global sites, he said.
Stilla hopes to further encourage decentralization of testing in the future, "moving away from the centralized model … and returning the power and the autonomy of testing to medical centers and reference labs."
And, Mourere said that the work of Quillien and her team at Eugène Marquis Cancer Center is hopefully a harbinger of a future where clinicians are "practicing real-time oncology," such that rather than waiting weeks for test results to change from the standard of care, patients can be switched "on the spot, when it matters."