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Biofidelity Prepares to Launch Lung Cancer Mutation Detection Assay

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NEW YORK – UK cancer molecular diagnostics company Biofidelity plans to launch its pyrophosphorolysis-based lung cancer mutation detection assay within the next few months.

Although the assay, called ASPYRE-Lung for Allele-Specific Pyrophosphorolysis Reaction, will initially be offered as a lab-developed test, or LDT, at the company's CLIA-certified laboratory in North Carolina, which opened last summer, Biofidelity's ultimate goal is for clients to conduct the assay themselves in a point-of-care setting.

ASPYRE-Lung can detect 111 mutations relevant to non-small cell lung carcinoma from across 11 genes using pyrophosphorolysis, a biochemical reaction that essentially works like reverse DNA synthesis.

In this reaction, a high concentration of pyrophosphate ions drives enzymatic DNA polymerization in reverse, resulting in the digestion of one DNA strand in double-stranded DNA. The reaction is almost entirely inhibited by the presence of even a single mismatched nucleotide pair.

To adapt this reaction to mutation detection, barcoded oligonucleotide probes are hybridized to target sequences. Each probe is matched to a mutation and therefore mismatched to wild-type sequences. When a mutation is present, the pyrophosphorolysis reaction digests away the perfectly matched sequences, releasing their associated barcoded probe tail, whereas the reaction stops at the point of mismatch on other sequences.

The free probe tails are circularized and amplified, with the barcodes revealing which mutations are present in a given sample.

As the reaction works with isolated DNA, it can be used with both tissue and blood samples.

Last year, Biofidelity published a research paper in Scientific Reports demonstrating this technique and showing 100 percent specificity and sensitivity, as well as an estimated false-negative rate of 1 percent in detecting specific substitution, insertion, and fusion variants.

Although targeted NGS assays have become standard in the cancer mutation diagnostic arena, ASPYRE-Lung might offer some advantages, including simplicity, low cost, high sensitivity, and a fast turnaround time.

"I would say the number one key thing is simplicity," said Barnaby Balmforth, Biofidelity's CEO.

For a start, the ASPYRE-Lung assay, though not based on PCR, runs on a real-time PCR instrument and doesn't require a complex next-gen sequencer.

"You can take our reagents, and you can take a real-time PCR instrument, and you can run those reagents on that instrument, and that's all you really need," Balmforth said. "You don't need to bring in new staff. You don't need to train them. You don't need informatics, and that's where the costs fall away."

Although Balmforth did not specify ASPYRE's cost, he maintained that it reaches a fraction of that of an NGS-based assay.

Researchers have been investigating pyrophosphorolysis as a diagnostic tool for some time but ASPYRE looks to be the first adaptation to succeed in becoming a commercial product.

Pyrophosphorolysis was discovered nearly 20 years ago. A technique similar to ASPYRE, called pyrophosphorolysis-activated polymerization, or PAP — a bidirectional reaction in which pyrophosphorolysis precedes primer polymerization — has also been investigated as a way to detect and monitor cancer mutations, but researchers have yet to find a way to perform it in a scalable and cost-effective manner.

"[It is] difficult to implement in a clinical setting except for very specific questions," Marc-Henri Stern, a researcher at the Institute Curie in Paris, who has used pyrophosphorolysis in detecting circulating tumor DNA, said via email. "The simplicity of NGS and its cost continuously decreasing [are] not favorable for the emergence of PAP."

Although Stern remarked that the sensitivity and specificity of pyrophosphorolysis in general are "outstanding," he cautioned that some mutations are challenging to target due to the complexity of their surrounding DNA sequences.

In designing ASPYRE, Biofidelity has overcome these obstacles largely by adopting a new approach to performing the technique.

Biofidelity's approach fundamentally differs from PAP by amplifying the probe, rather than the sample DNA, following pyrophosphorolysis.

"We're using this pyrophosphorolysis reaction to activate a probe, which itself is then amplified and detected," Balmforth explained. "And while that may seem like a very subtle difference, that actually unlocks this potential to have exquisitely high sensitivity, but to be able to scale it to large numbers of genomic variants."

This also helps the company circumvent some of the past challenges involved in primer design. Past approaches have tended to design PCR primers that must hybridize precisely to certain positions to be activated and amplified, requiring very high probe purity to avoid off-target interactions. Biofidelity, in contrast, makes probes that more broadly cover regions of interest, providing greater flexibility in probe design.

"That means if there are challenging sequences, you can design multiple different probes and find the ones that work, and we've yet to find any sequences where we can't make a probe work," Balmforth said.

Importantly, ASPYRE-Lung can be used to analyze both DNA and RNA with the same set of reagents.

"That gives you a lot of flexibility in how you build out panels to cover large numbers of variants," Balmforth said, noting that fusions are an area where NGS tends to struggle. "To be able to combine those into a single test gives us some really significant advantages."

Biofidelity is currently working with research partners to beta-test the company's ASPYRE reagents. The company plans to launch ASPYRE-Lung as an LDT at its North Carolina facility within the next few months, with a full commercial launch later this year.

ASPYRE-Lung is the first of a portfolio of products targeting other cancer indications that Biofidelity is pursuing, all based on its ASPYRE technology.

"We prioritize where we think the technology will have the biggest possible impact on patients," Balmforth said.

Future indications under review include colorectal cancer, pancreatic cancer, melanoma, and hematological cancers. Although the order in which those products will be prioritized remains to be decided, the company does expect to announce a second product later this year.

In advance of its launch, Biofidelity is building up its US workforce. The company currently employs about 10 people in the US and plans to expand to more than 30. Biofidelity is currently hiring for positions in business development and jobs at its North Carolina lab.

While ASPYRE-Lung will launch first in the US, Biofidelity plans to commercialize it abroad later.

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