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Screening Pipeline Yields Methylation-Based Marker Candidate in Ovarian Cancer

By Andrea Anderson

NEW YORK (GenomeWeb News) – In PLoS ONE last night, American, Austrian, and British researchers described the approach that they are using to find potential methylation-based markers for detecting ovarian cancer DNA in the blood.

The team relied on array-based screening to glean methylation differences between dozens of ovarian tumors compared to two normal blood cell samples from unaffected, age-matched individuals. They then took markers that appeared to be tumor-specific in that discovery stage on to verification testing.

The search led to a site in the IFFO1 promoter that was typically methylated in DNA from ovarian cancers. Subsequent experiments on blood samples that had been collected from women before and after surgical removal of their tumors offered hints that IFFO1 methylation status may augment existing methods for tracking ovarian cancer progression and recurrence.

"We anticipate that [IFFO1 methylation] and other candidate markers emerging from this marker development pipeline may provide disease detection capabilities that complement existing biomarkers," University of Southern California researcher Peter Laird, the study's senior author, and his colleagues wrote.

Because it is largely asymptomatic, ovarian cancer is often not detected until it has metastasized to other parts of the body, the study authors explained. Although patients diagnosed at such advanced stages may respond to therapy at first, they often relapse following this treatment.

"It's detected, in general, when it's too late," the study's co-first author Mihaela Campan, a research associate in Laird's USC lab, told GenomeWeb Daily News. "There's a definite need to get markers that will help us either detect the disease early or help us decide on the therapy."

Levels of two proteins — CA-125 and HE4 — can provide information on disease status for individuals already diagnosed with ovarian cancer, she explained, but are less useful for detecting the disease or predicting risk since they are not found at high levels in all ovarian cancers.

"The protein markers are not specific enough for early detection of the cancer," Campan said. "For that reason, we can only use them to monitor recurrence."

To try to track down DNA methylation signals coinciding with the disease, the team screened 41 ovarian tumor samples collected at the University of Southern California and Duke University Medical Centers, using Illumina's Infinium HumanMethylation27 BeadArray platform to gauge the methylation at more than 27,500 CpGs.

Rather than comparing the tumor methylation patterns with those in non-cancerous ovarian tissue, the team focused on finding sites that consistently showed differential methylation in the tumor and normal blood serum or blood cell samples.

"Since we are doing blood-based detection," Campan explained, "the most important thing was that this marker is not present in the blood of normal people."

For the screening stage of the study, the researchers compared the 41 tumor samples with peripheral blood leukocytes from two post-menopausal women without ovarian cancer.

Following steps to weed out problematic methylation probes and sites showing higher methylation in blood than in tumor samples, they were left with 15 of the most promising methylation marker candidates.

They whittled this set down even further through comparisons to ovarian cancer methylation data from The Cancer Genome Atlas data portal. The team also tossed out candidates that were methylated in 10 normal blood plasma samples or in two normal peripheral blood leukocyte samples tested by sensitive, PCR-based methylation analyses.

At the end of the verification testing, the team was left with a single candidate marker: a methylation signal in the IFFO1 promoter that was far more common in tumor samples than in the blood.

The same methylation marker could be found in blood samples from women with ovarian cancer, too, they found. Using a digital PCR-based methylation detection method, the researchers tested for IFFO1 methylation, or IFFO1-M, in 127 blood samples collected from 16 ovarian cancer patients at various time points after tumor resection.

Although some of the samples were decades old, Campan explained, the team was still able to get DNA and assess IFFO1 methylation in most of the samples.

Prior to surgery, IFFO1-M was high in blood samples for eight of the women. Samples from these patients were included in the researchers' longitudinal study comparing the IFFO1-M patterns in the blood with levels of the protein biomarker CA-125 over time after surgical removal of the tumors. One patient whose CA-125 levels were negative at the earliest time point was also included the longitudinal study.

Six of the nine individuals had IFFO1-M profiles that significantly correlated with blood CA-125 levels over time, they found. Both IFFO1-M and CA-125 levels typically dropped off following surgery but went up again over time in individuals who experienced ovarian cancer recurrence.

Folding in information on IFFO1-M status also slightly improved the number of ovarian cancer recurrences that could be detected, they reported: CA-125 levels corresponded with recurrence in blood samples from six of the eight women who had had disease relapse in the longitudinal study. Meanwhile, IFFO1-M patterns coincided with four of the recurrent cases, including one case that would have been missed by looking at CA-125 levels alone.

"There's always a need to get better markers for recurrence," Campan said. "Our marker, at least, can compensate for the cases where it is not picked up by the [CA-125] protein marker."

The researchers plan to do additional studies looking at the utility of IFFO1-M for diagnosing ovarian cancer in blood samples of women suspected of having the disease, Campan explained. They are also following up on other DNA methylation marker candidates in ovarian cancer and using the same screening pipeline to look for methylation-based markers in other cancer types.

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