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Ovarian Cancer Test Based on Engineered Toxin Developed by Australian Team

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NEW YORK (360Dx) – Australian researchers are developing a blood-based platform that has shown success in a preliminary study in detecting early-stage ovarian cancer.

In a study published recently in Biochemical and Biophysical Research Communications and involving the analysis of 69 serum samples, researchers at Griffith University and the University of Adelaide said serum from patients with all stages of ovarian cancer had elevated levels of N-glycolylneuraminic acid (Neu5Gc) — an abnormal glycan, or sugar, expressed on the surface of human tumor cells and released into the blood.

The researchers used surface plasmon resonance to detect Neu5Gc glycans using SubB2M, a bacterial toxin that binds to the glycan and that they have engineered for enhanced sensitivity and specificity in blood and tissue samples. SubB2M is the B subunit of the subtilase cytotoxin produced by Shiga toxigenic Escherichia coli.

The developers anticipate that clinicians could eventually use the Neu5Gc biomarker for diagnostic purposes, but they will likely need to prove its clinical utility on a large volume of blood samples before getting regulatory approvals.

They are seeking a commercial partner to help them develop a diagnostic test that, if successful, could be made available to physicians for use in broad screening of cancers, including early-stage ovarian cancer. Within about two years, if the group's collaboration and the R&D initiatives are successful, the platform could become part of routine testing that people receive each year during an annual physical examination, James Paton, a professor of microbiology at the University of Adelaide and study author, said in an interview.

When a microliter of serum is passed over the surface of a microchip bound with the bacterial toxin, glycoproteins that consist of N-glycolylneuraminic acid adhere to the chip, changing the system's surface resonance so that it is detected by a sensor.

The blood test would be used to rule in or rule out the presence of cancer, and patients with positive results would then receive additional testing to confirm the condition and provide more clinical detail, Paton said.

"This [study] clearly demonstrates that levels of Neu5Gc can be detected in sera of patients at different stages of ovarian cancer," Jean-François Masson, a professor of chemistry at Université de Montréal not involved in the current study, said in an interview. "This is an important step forward as it constitutes the first report in the scientific literature for the detection of this class of molecules in sera of patients with surface plasmon resonance technology."

Early detection

Early detection of ovarian cancer can be crucial to patients' survival, Paton said. The five-year survival rate for ovarian cancer is less than 20 percent in women diagnosed at late stages but that figure increases to more than 90 percent when it's detected in stages I and II.

Biomarkers other than Neu5Gc have shown some promise as diagnostic tools. Despite recognized limitations, CA125 is among the most widely studied serum biomarker for ovarian cancer. It is FDA approved for disease monitoring in patients diagnosed with epithelial ovarian cancer and used in combination with transvaginal ultrasound to screen high risk women, including those who are known to harbor BRCA1 mutations.

In March, diagnostics firm Vermillion said that it anticipated launching a study in Israel to explore use of its OVA1 and Overa tests in place of the established ovarian cancer marker CA125 for monitoring patients with BRCA1/2 mutations who are at high risk of developing the disease. The firm's OVA1 serum-based test, which combines the results of five immunoassays into a single numerical score, has been FDA cleared to help assess the likelihood that malignancy is present when the physician’s independent clinical and radiological evaluation does not indicate malignancy, but it is not intended as a screening or stand-alone diagnostic assay.

Studies from the US and UK researchers have found that diagnostics firm Abcodia's ovarian cancer test could be useful for monitoring patients at high-risk of developing the disease. Harvard Medical School researchers working with Abcodia are looking at whether adding markers beyond CA125 might improve the performance of their assay.

In their study, the Australian researchers noted that CA125 serum levels can be elevated in patients with ovarian cancer, but it also can be elevated in nonmalignant conditions such as endometriosis, pregnancy, ovarian cysts, pelvic inflammatory disease, hepatitis, cirrhosis, and in the follicular phase of the menstrual cycle.

Paton said that it is feasible to investigate combining CA125 as a biomarker along with N-glycolylneuraminic acid in helping determine if a patient has ovarian cancer. But CA125 is not a reliable marker alone because its levels are very low, he said.

His team used a CA125 ELISA assay on the serum samples that it had tested for N-glycolylneuraminic acid. They found that CA125 was absent in samples from patients with stage I ovarian cancer but there were detectable levels of CA125 in about 10 percent of the samples from patients with stage II cancer. CA125 was detectable in about 20 percent of samples from stages III and IV cancer patients.

"We found that when we used SPR to detect the toxin, about 90 percent of serum samples from women with stage I ovarian cancer had elevated levels of N-glycolylneuraminic acid, and 100 percent of serum samples form women with stages III and IV cancers had elevated levels," Paton said.

Biomarkers needed

Women with ovarian cancer are usually diagnosed in the late stages due to the largely asymptomatic nature of the disease and the lack of highly sensitive and specific biomarkers. Ovarian cancer, which kills about 150,000 women each year, has the highest mortality rate of the female reproductive cancers.

Earlier this year, the Australian researchers published the results of a study in BBRC that described the specificity and utility of SubB2M, noting that they anticipated the biomarker could be useful for the testing of serum and other bodily fluids for cancer diagnosis and prognosis.

Paton said that the assay is highly sensitive and specific because its engineered toxin binds to N-glycolylneuraminic acid, but not to an inactive N-acetylneuraminic acid, an enzyme expressed by healthy humans that would normally generate background noise that interferes with the system's performance. "We have increased its affinity for N-glycolylneuraminic acid and eliminated any background binding to N-acetylneuraminic acid so that we have a more specific and discriminatory binding agent," he said.

In addition to ovarian cancer, Paton and his colleagues are investigating the use of Neu5Gc to detect breast cancer, and he noted that the group anticipates that the biomarker could be implemented as part of a blood test that detects many cancers.

The blood biomarker could also be used alongside traditional markers for cancers, and its diagnostic chip could be incorporated into different types of diagnostic platforms, such as immunohistochemistry, molecular, or sequencing platforms, Paton said.

The use of SPR technology can speed up assay times for the detection of cancer markers, said Masson, who wrote a review of SPR's use in medical diagnostics published last year in ACS Sensors.

While the technology is being applied increasingly in the development of tests for a series of cancer markers, none have been approved yet by regulatory agencies, he noted.

A logical progression for the authors of the recent study is to now focus on increasing the number of samples tested with the technology, Masson said.

The Australian group has been testing serum samples obtained from the Victorian Cancer Biobank and is seeking funding to test a larger number of samples — possibly as many as 1,000 according to Paton — so that they can establish thresholds for healthy individuals and determine normal diagnostic ranges.