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Johns Hopkins Team to Commercialize Barrett's Esophagus Epigenetic Biomarker Panel

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NEW YORK (GenomeWeb) – A team of researchers led by the Johns Hopkins University of Medicine has developed a methylated epigenetic biomarker panel integrating a sponge-based collection tool and a methylated-bead PCR method that it believes can detect Barrett's esophagus (BE).

The group is now working with the university to commercialize the panel as a diagnostic assay to diagnose patients with BE — especially in asymptomatic individuals — and potentially prevent esophageal adenocarcinoma (EAC).

BE occurs when mucosal cells lining the lower section of the esophagus abnormally develop in patients. Clinicians only detect BE by using invasive endoscopy and examining tissue from the area with a tissue biopsy. However, asymptomatic BE often goes undiagnosed and lead to EAC in certain patients.

"Endoscopy can't be performed on everybody because it's just too expensive and perhaps somewhat risky and inconvenient," explained Johns Hopkins gastroenterology and hepatology professor Stephen Meltzer, who was the senior author of the study. As a result, "the vast majority of Barrett's patients are not diagnosed because they don't get endoscopies."

In the study, published last month in Clinical Cancer Research, the researchers examined the safety, feasibility, and efficacy of a sponge cytology-sampling device, called the EsophaCap, with eight previously identified BE biomarkers.

Using the EsophaCap tool, the team looked at two separate patient groups with cytologic samples from 80 individuals who had previously undergone upper endoscopy (52 in the training set and 28 in the test set). In the training set, 18 had BE while 34 were controls. In the test set, 14 individuals had BE while the other 14 had gastrointestinal symptoms without BE.

During each collection cycle using the EsophaCap, the patient swallowed a small capsule while the researcher held onto the end of the filament, which is attached to the capsule with the string. Once inside the stomach, the capsule dissolved after several minutes and allowed a 2 centimeter-sized polyurethane sponge to expand.

After waiting a few minutes, the physician gently tugged the string dangling outside the patient's mouth. As the sponge passed through the esophagus and out the patient's mouth, it collected cytologic material. According to Meltzer, the EsophaCap tool collects about 500,000 cells per swab.

Meltzer and his team then applied a technique called methylation on beads (MOB), developed by fellow senior author Jeff Wang. The method allows researchers to perform capture, retention, and bisulfite conversion of small quantities of DNA. Meltzer noted that the tool uses superparamagnetic beads to extract and increase the efficiency of the patient's methylated DNA with minimal impurities.

"With MOB, you do the bisulfite treatment and the DNA extraction in one tube, [where] you put up an actual physical permanent magnet on the side of the tube, [you] gather all the beads and hold them inside the tube," Meltzer explained. "You can pour off the remaining liquid, and you now have concentrated beads with all the DNA that's already bisulfite treated."

The team then performed real-time quantitative methylation-specific PCR to measure target gene methylation levels in bisulfite-converted DNA obtained from EsophaCap samples. They found that five biomarkers — p16, HPP1, NELL1, TAC1, AKAP12 — were significantly higher in the DNA of BE patients than in control patients.

Afterward, the team used a lasso regression analysis to determine a prediction model for the genes, and selected four biomarkers — p16, NELL1, TAC1, AKAP12 — as the most efficient to use to develop an epigenetic panel.

According to Meltzer, the overall EsophaCap sample collection process only takes about four to five minutes. In the study, he noted that about 85 percent of patients successfully swallowed the capsule with complete extraction success. After placing the sample into methylated preservatives, the researchers only required one to two hours to produce a result.

The study's training set had a clinical sensitivity of 95 percent and specificity of 62 percent, while the test set had a sensitivity of 79 percent and specificity of 93 percent. Meltzer attributed the discrepancy in the data to the study's small population and said that the team will improve the results by collecting more patients in future studies that include a multicenter cohort.

Meltzer also acknowledged that his team dealt with issues regarding adequate sample collection, as some patients were unable to swallow the capsule. Because the capsule has a string attached to it, some patients felt strange swallowing the capsule and exhibited a belching reflex, as the sensation of tugging the capsule back up can feel "unnatural since the string's going in the wrong direction."

However, Meltzer highlighted that his team did not find any internal esophageal scarring, bleeding, or trauma from the sponge or the capsule on patients.

"One of the added benefits … is that the higher the methylation level, the closer the malignancy the Barrett's is," Meltzer noted. "We want to do another follow-up study in cancer and dysplasia to show that we can actually distinguish between cancer, dysplasia, and benign Barrett's patients."

Meltzer said that his team has partnered with Johns Hopkins to commercialize an epigenetic biomarker panel based on the four genes the team identified in the lasso regression model. His team aims to launch a company with the technology licensed from the university.

The group plans to launch the epigenetic assay as a laboratory-developed test called "EsoBee", which he said could potentially segue into a kit. Meltzer and his team recently received a US patent for the assay.  

As Meltzer and his team commercialize a biomarker panel integrating EsophaCap and MOB, companies and other research teams are also developing their own methods to detect BE in patients at risk for esophageal cancer.

Medtronic has developed a similar tool called CytoSponge, which is a small mesh sponge tool within a soluble gelatin capsule that patients swallow. The firm then searches for TP53 mutations in the mucus samples, which the current study authors noted had a 73 percent sensitivity and 94 percent specificity in detecting BE in a previous study.

In addition, PavMed spinoff Lucid Diagnostics has partnered with Case Western Research University to develop an assay called EsoCheckRapid, which is a squamous cell-based method that detects several BE biomarkers, including CCNA1. The firm's goal is to receive both US Food and Drug Administration 510(k) approval for the EsoCheck device and CLIA/CAP approval for its reference laboratory this year.  

Lucid Dx CEO and Chairman Lishan Aklog, who is not affiliated with Johns Hopkins, said that that team of researchers will need to take care of certain issues as they move forward developing the technology. Importantly, he noted that the researchers will need to ensure the biomarker panel maintains high sensitivity and specificity before extrapolating the results to a larger patient population.

Aklog pointed out that significant age gap between the team's test cases and controls may influence the results of regression models for predicting accuracy, as the patients in the study's case group were much younger than the test group's patients.

"You worry that some of the impact of age in the assay is going to be challenged, since most patients that are candidates are going to be over 50, so there's less age dependence over 50," Aklog said. "If you have cases and controls that don't match for age, and then if you use age as a discriminant … [its] something we should keep an eye on in larger cohorts."

Aklog also noted that the team may have issues collecting accurate samples during each extraction process, as the sponge-based method collects cells from the entire esophagus.

In contrast, Lucid Dx's EsoCheck collection method uses inflatable balloon capture technology that Aklog believes delivers more targeted results. After the capsule arrives in the stomach, the balloon inflates and is gently pulled through the lower esophagus. Once the nurse feels the esophagus tighten around the balloon, they pop it with a small syringe. The balloon shrinks and is contained within the capsule, which Aklog emphasized helps collect a targeted sample because the capsule prevents contamination as it travels out of the esophagus and through the patient's mouth.

"It's well known that the cells in the upper esophagus are subject to methylation, particularly in smokers," Aklog said. "So you can certainly get false positive samples by having the lower esophageal cells contaminated by the proximal cells, which are not susceptible to not getting Barrett's."  

In addition, Aklog said that the Hopkins team's use of quantitative PCR to detect methylated biomarkers may also encounter noise issues as it collects samples from larger patient populations. In contrast, Lucid Dx's tool uses next-generation sequencing, which Aklog believes will be less prone to errors than quantitative PCR.

In general, Aklog believes the market for BE-based diagnostic assays will expand rapidly in the next few years and that there will be more players in the space overall as several academic groups are searching for methylation biomarkers that indicate the presence of BE.

"There is clearly an unmet need and patients who are candidates for BE, [and] there's been lots of activity screening dozens, hundreds, even thousands of genes to look for correlation with BE," Aklog explained. "If [patients] are detected to have BE, then they have an option of close surveillance, and if they progress to dysplastic conditions, intervention."

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