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Circulogene Publishes First Patient Data From Liquid Biopsy Test

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NEW YORK (GenomeWeb) – Liquid biopsy firm Circulogene has published the first patient data showing that its liquid biopsy test is concordant with tissue-based biopsy analysis and may be able to detect recurrence earlier than standard methods.

Although the numbers are small — the company published a case report of two patients in the journal Cancer Therapy & Oncology and presented an abstract earlier this month at the American Society for Clinical Oncology meeting detailing results in 14 patients —the assay detected more mutations per patient and in more genes per patient than analyzing DNA from a tissue biopsy, Circulogene CSO Chen-Hsiung Yeh told GenomeWeb.

Circulogene first launched its assay, which uses finger-stick volume blood samples and next-generation sequencing, to analyze circulating tumor DNA, last year out of its CLIA-certified laboratory in Birmingham, Alabama. Since then, the company has analyzed more than 1,000 patient samples, Yeh said.

It seeks to distinguish itself from other liquid biopsy companies by requiring much smaller sample volumes for testing. Yeh said the company is able to do this because it has developed proprietary sample prep technology that does not rely on DNA extraction, isolation, and purification — protocols that end up losing about 70 percent of the starting material.

Yeh declined to disclose the methods of the technology, but the firm has previously said it is based on methods that were originally developed by the company's founders while they were at cardiovascular testing company Atherotech Diagnostics. After starting Circulogene, the group developed ctDNA isolation technology that uses fractionation and enzyme manipulation — now the core of its liquid biopsy assay.

Circulogene's ctDNA test relies on Thermo Fisher Scientific's Ion AmpliSeq technology and the Ion Proton to sequence around 3,000 mutations in 50 cancer-related genes. In the study presented at ASCO, the company collaborated with researchers at the University of Alabama, Birmingham to compare performance on cell-free DNA with DNA extracted from a tissue biopsy in 14 ovarian cancer patients. From each patient, the team analyzed samples both before and after treatment with neoadjuvant chemotherapy.

Overall, the researchers found that the ctDNA assay identified more mutations in more genes than the tissue-based assay. One reason for this, said Yeh, could be the inherent sampling bias in tissue biopsies. "You don't know how many tumor cells you'll get from a biopsy," he said. In addition, tumors themselves are heterogeneous, so a different location on the tumor may contain different mutations.  

The researchers also noted that liquid biopsies may be analyzing the genomes of dying tumor cells, which could be different from the remaining tumor cells that are analyzed by tissue biopsy.

Prior to the patients receiving chemotherapy, the ctDNA assay found 57 mutations in 19 genes, while the tissue-based assay identified 38 mutations in six genes. In addition, 14 of the 19 mutated genes are associated with approved targeted therapies.

While the ctDNA assay identified more mutations, Yeh said that overall, the results were concordant. The ctDNA assay picked up the same mutations as the tissue assay.

Analyzing the patients after therapy, the researchers found 54 mutations in 21 genes from the ctDNA assay compared to 37 mutations in six genes from tissue-based analysis. In addition, the mutations and mutated genes were more likely to change between pre- and post-treatment when analyzing ctDNA versus DNA from a biopsy.

Of the 57 mutations identified in the ctDNA of pre-treatment samples, only six persisted after treatment. By contrast, of the 38 mutations identified in the tumor, 33 persisted following treatment.

Separately, the company recently published a case report of two patients — one with metastatic lung cancer and another with metastatic peri-pancreatic lymph node adenocarcinoma with unknown primary — in collaboration with clinicians at Hope Health Center in Reseda, California.

In this study, the researchers used the liquid biopsy test to monitor the patients' tumor burden and treatment response, running the assay along with conventional follow-up tests such as computed tomography, positron emission tomography imaging, and analysis of protein biomarkers.

The lung cancer patient, a 69-year-old non-smoking female, had an EGFR mutation and was ALK, ROS-1, and BRAF negative, so was placed on Tarceva and Avastin. After five months of therapy, protein biomarkers dropped and CT/PET scans showed that her tumor had stopped growing and had even begun to shrink.

The clinicians continued to monitor the patient's protein biomarkers via an immunoassay and mutational burden via the ctDNA assay. Although the immunoassay continued to show low biomarker levels, the liquid biopsy detected the emergence of new mutations in TP53 and PTEN. Additional analysis identified low-frequency subclonal mutations in EGFR, indicating selective pressure by the drugs.

A few months after the first PET and CT scans, new ones did indeed show a new tumor mass, "confirming what the ctDNA mutation analysis had found earlier," the authors wrote.

In the second patient, the researchers found that the liquid biopsy assay correlated well with other clinical findings. At treatment, ctDNA analysis identified four mutations, which dropped throughout the course of the patient's treatment. Additionally, PET and CT scans also showed a significant decrease in tumor size with a more than 90 percent response rate to the treatment.

"The mutation detection of ctDNA in drops of blood is a powerful monitoring tool capable of providing accurate and earlier assessment of tumor behavior, burden, and patient responses following treatment," the authors wrote.

Yeh said that Circulogene plans to continue to validate its assay with both academic and clinical partners, and also plans to apply for grants to fund clinical studies.

Circulogene is a newcomer to the rapidly growing liquid biopsy space. Nonetheless, Yeh said that it is not necessarily looking to compete with other firms including Guardant Health, Foundation Medicine, and recent startups like Inivata and AccuraGen. Instead, he said, Circulogene's technology could actually be compatible with other liquid biopsy techniques since the company focuses on the front-end portion of the assay.

While other companies have been developing techniques to push the limit of detection to ever-lower frequencies, Circulogene has focused on the very first steps of isolating the cell-free DNA.

"Our ultimate goal is to provide a front-end solution for cell-free DNA and cell-free RNA analysis from any body fluid, including plasma, serum, urine, cerebrospinal fluid, and eventually saliva," Yeh said.

In addition, Yeh said that Circulogene's technology could have applications in other fields including noninvasive prenatal technology, organ transplantation, infectious disease, and even biodefense.

For instance, he said, one potential biodefense application is to triage wounded soldiers, he said. There is some evidence indicating that higher levels of circulating cell-free DNA are associated with a more severe trauma or burns, he said. Because Circulogene's technology relies on small amounts of blood, cell-free DNA analysis could eventually be done in the field on portable devices, Yeh said. However, such applications are further down the road.

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