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EZLife, UCLA Team Aim to Expand EGFR Lung Cancer Assay Into RUO Offering


NEW YORK (GenomeWeb) – After recently winning a $5 million grant from the National Cancer Institute, a cross-department consortium of researchers at the University of California, Los Angeles are now developing an expanded EGFR biomarker assay based on the group's electric field-induced release and measurement (EFIRM) platform.

The consortium — which includes UCLA's School of Dentistry and pathology, pulmonology, and radiology departments — has partnered with EZLife Bio, a Los Angeles-based startup founded in 2016 by UCLA professor Wei Liao. The partners aim to enter the lung cancer diagnostic market with the assay for the early detection of forms of non-small-cell lung-carcinoma (NSCLC).

In order to detect early stages of lung cancer, researchers often use low-dose computed tomography (CT) to screen patients with high risks of developing the disease. However, clinicians struggle to accurately detect cases of lung cancer due to high false-positive rates, which can create unnecessarily high costs and other dangers for patients.

"One of the main obstacles is that CT has a low sensitivity, which is an issue when we identify a 'strange' nodule' in a person's lung," EZLife Bio Director of Research and Development Michael Tu explained. "We can only do something called 'watchful waiting' if it's a small nodule, which can cause patient anxiety and stress."

If a patient is a high-risk individual, such as an elderly patient who has smoked a pack of cigarettes a day for 30 years, Tu believes the EGFR panel may help confirm if the nodule identified in CT scans is cancerous or benign in nature.

Tu explained that Liao originally developed the EFIRM technology while working as a senior researcher under associate professor David Wong at the UCLA School of Dentistry. According to Tu, the team conceptualized the idea of using EFIRM as a companion molecular diagnostic tool to load spiral CT scans.

EFIRM uses a conducting polymer-based electrochemical chip with electrodes, applying electric fields to enhance hybridization efficiency and identify specific duplexes. A capture probe precoated with conducting polymer measures EGFR sequences by immobilizing DNA fragments on the electrode and using the electric fields to manipulate the genetic material's movement, which enhances hybridization. Tu noted that the platform's electric field can also lyse open exosomes that may contain additional mutated genetic material.

Tu highlighted that the EGFR panel only requires 20 microliters of a person's saliva or blood, and that the assay can detect EGFR mutations in less than three hours. The panel can be run on 96-well plates, allowing researchers to simultaneously multiplex several samples. EZLife currently has CE marking for a benchtop reader that processes the plates.

Tu said that researchers will be able to purchase the instruments and reagents for research use only, and that the firm hopes to soon offer the EFIRM lung cancer assay as a service.

In a study published earlier this month in the Journal of Molecular Diagnostics, Liao and his team wanted to see if they could use EFIRM technology to accurately detect two EGFR mutations — p.L858R and exon19 deletion — in patients with early stage NSCLC.

The team recruited 248 patients with radiographically determined pulmonary nodules, collecting plasma before performing biopsy and histologic examination on the samples.

The team established an inclusion criteria of benign nodule (control) and stage I or II lung cancer that had either EGFR mutation. Excluding 152 patients because they were diagnosed with advanced stage lung cancer, the team used plasma samples from 44 patients, 23 of which had biopsy-proven benign pulmonary nodules, and 21 of which had stage I or II lung cancer. Liao and his team then analyzed the samples for EGFR mutations using EFIRM.

The researchers found a sensitivity of 92 percent for the p.L858R mutation and 77 percent for exon 19 deletions. In addition, they found a clinical specificity 92 percent. They encountered two false-positive results in 23 patients with EGFR-positive nodules, and therefore a 95 percent specificity for the entire series.

Acknowledging the difference in sensitivity between the two mutations, the researchers believe that biological variation in the amount of cfDNA in certain patients may have skewed the data. In addition, they noted that the issue may be caused by a "true technical difference between the ability of the capture and signal probes to detect small amounts of circulating DNA."

However, the team also noted that the sensitivity of EFIRM is limited by the percentage of tumors that contain either or both of the variants in the assay.

Importantly, the team found that the tool maintained perfect concordance with identical mutations discovered in plasma and nodule biopsies.

Tu explained that EZLife Bio is in the process of negotiating a license with UCLA for several patent filings surrounding the technology. EZLife Bio plans to offer the instruments and disposables for the assay, as well as eventually implementing the process on an automated platform Tu said that the firm is also filing additional patents independent of the UCLA group.

"EZLife Bio is providing the platform associated with the technology ... a tool that Wong can use as part of his molecular and clinical research at UCLA," Tu said. "We are more focused on the instrumentation side of things because we want to develop a tool that people can use to deploy the EFIRM technology."

While the current EGFR panel currently measures L858R, exon 19 deletion, and T790 mutations, Tu noted that the $5 million NCI grant will help the consortium develop and validate independent assays for seven additional mutations, including variants in KRAS, PIK3CA, and TP53.

"We're expanding work into other mutations that may be relevant," Tu said. "We want to increase how wide of a net we're casting to increase the [panel's] positive predictive value."

However, Tu acknowledged that adding the other biomarkers to the EGFR panel will pose a challenge because the consortium will need to secure clinical resources to develop the technology, in addition to collecting samples for clinical work.

Tu also pointed out that "development, initial proof concept, and validation will require a longer period of time, since many of the mutations will have higher complexity than we thought."

The study authors envision that an expanded 10-biomarker panel covering mutations expressed in half of all lung malignant tumors could "be used to establish the clinical utility for screening patients with indeterminate pulmonary nodules."

Since its founding in 2016, EZLife Bio has raised about $10 million in funding from angel and venture capital investors. However, Tu noted that much of the funding has been from investors in East Asia, and that the company may eventually pursue fundraising in the US as it finalizes the IP situation.

According to Tu, EZLife Bio is targeting the in vitro diagnostic and research markets with the EGFR assay. The firm is also exploring potential life sciences and research-use-only applications as it seeks to achieve CLIA approval and LDT status for its assay.

Other groups have been developing electrochemical-based tools to detect a variety of cancers. A team of researchers at the University of Toronto led by biochemistry professor Shana Kelley is developing its own electrochemical chip method to detect both tumor RNA molecules and DNA sequences without enzymatic amplification.

University of California, San Diego startup Biological Dynamics is developing an electrochemical chip platform that uses alternating current to isolate cf-DNA, proteins, and exosomes. The group published data earlier this year demonstrating that the chips can be used to rapidly isolate exosomes from plasma or whole blood samples to detect cancer biomarkers.

Kelley highlighted that electrochemical tools can be highly multiplexed, allowing researchers to build out large panels to detect a multitude of genetic mutations.

"The key thing about these types of applications is that you need high levels of both sensitivity and specificity, which the [EGFR panel] seems to have," Kelley noted.

Importantly, Kelley argued that the clinical space needs "these types of low-cost portable tools that can be used at the point-of-care space. Right now, the healthcare system is okay with paying for expensive [sequencing] approaches, but we will have to eventually gravitate to cheaper systems, where electrochemical tools will play a role."

At the same time, Kelly acknowledged that there aren't many players yet in electrochemical-based molecular detection because most groups have not achieved a level of clinical sensitivity and specificity performance that matches sequencing approaches. Electrochemical tools like EFIRM will need to offer "clearly defined benefits" to distinguish themselves from sequencing options to look at genetic mutations.

In addition to ctDNA, Tu noted that the EFIRM platform can simultaneously examine other molecular targets, including miRNA and protein biomarkers. With the NCI funding, the consortium will introduce a six-biomarker panel of miRNA to compliment and strengthen the lung cancer panel.