Ovarian Cancer Biomarker Search Uncovers Low-Frequency TP53 Mutations in Healthy Individuals

Ovarian Cancer Biomarker Search Uncovers Low-Frequency TP53 Mutations in Healthy IndividualsSomatic mutations in the cancer-related gene TP53 turn up at very low levels in peritoneal fluid from healthy individuals, becoming more prevalent in women with ovarian cancer, according to a study published this week in the Proceedings of the National Academy of Sciences.

Researchers from the University of Washington used a se nsitive method called duplex sequencing to search for low-frequency TP53 gene mutations in peritoneal fluid samples from 17 women with high-grade serous ovarian cancer (HGSOC) and 20 women without.

The team detected TP53 mutations in peritoneal samples from all but one of the women with HGSOC. But similar mutations also turned up in samples from 19 of the 20 unaffected control individuals, albeit at far lower frequencies. A series of follow-up experiments indicated that the overall burden of TP53 mutations in the peritoneal fluid could be used to distinguish between cases and controls with 82 percent sensitivity and 90 percent specificity.

“We think eventually it could be useful as a biomarker, based on the mutation load,” senior author Rosa Ana Risques, a pathology researcher at the University of Washington, told GenomeWeb.

“The mutation load [in TP53 or other cancer genes] could be a biomarker for cancer detection or prediction,” she added, explaining that additional research is needed to explore that possibility.

Risques noted that her team is generally interested in finding biomarkers for early cancer detection. For the latest study, they decided to take advantage of what she called “a really good technology and a really good model system to check it.”

The technology — duplex sequencing — is designed to overcome errors introduced during sequencing by profiling both strands of DNA to make sure that the same apparent mutation is present at the same site on each, she explained, making it possible to see low-frequency mutations that would otherwise be indistinguishable from sequencing errors.

“The error rate of standard next-gen sequencing is one in 1,000 nucleotides and for duplex sequencing it’s just one in 10 million,” Risques said.

By employing distinct and asymmetrical adaptor sequences to amplify DNA from each strand, the researchers effectively tagged each strand of DNA, making it possible to bioinformatically identify and correct errors in the sequences, while picking out true mutations, noted co-author Michael Schmitt, a pathology, hematology, and medical oncology researcher at the University of Washington who helped develop the duplex sequencing method.

For the current analysis, for example, the team used duplex sequencing to search for low-frequency somatic mutations in TP53, a driver gene that’s altered in almost all HGSOC tumors.

Using DNA isolated from peritoneal fluid samples, the researchers developed indexed duplex sequencing libraries and sequenced pooled samples on the Illumina HiSeq 2500 instrument, sequencing to a depth that allowed them to pick up a single TP53 mutation in 20,000 molecules.

“We are working with biopsies that have mostly normal cells and we want to find one cancer cell or a few cancer cells within these biopsies,” Risques said. “So we are trying to find that rare, mutated cell.”

Although peritoneal fluid is not easily accessible or collected for routine cancer screening, it is typically collected and inspected by pathologists for women with suspected ovarian cancers who undergo related surgical procedures, explained co-author Jeffrey Krimmel, also at the University of Washington.

Of the 17 HGSOC cases profiled, eight involved individuals with early-stage ovarian cancers. Seven individuals with ovarian cancer and 10 of the controls carried germline mutations in the breast and ovarian cancer risk genes BRCA1 or BRCA2.

The researchers uncovered low-frequency TP53 mutations in samples from 35 of the 37 cases and controls.

While these somatic changes in the gene appeared to be found at low levels in peritoneal fluid regardless of cancer diagnosis or BRCA mutation status, the frequency of these mutations was much lower in unaffected control individuals, affecting one in more than 13,000 copies of the gene, on average.

“This was a bit surprising at first, but is actually concordant with what the literature is showing,” Risques said, pointing to studies on aging blood cells profiled genomically in the past.

Similarly, when she and her colleagues used duplex sequencing to search for low-frequency TP53 in circulating white blood cells in peripheral blood from 15 women, they found low-frequency TP53 mutations across the board, regardless of ovarian cancer status.

“Now that we can go much, much deeper with duplex sequencing, we see that these mutations in cancer genes are actually carried by nearly everybody — they are at very low frequency, but they are there,” Risques said.

While the presence of these mutations could theoretically throw a wrench in plans to use somatic mutation status as a marker for cancer, the team believes that it should still be possible to differentiate between cases and controls based on mutation prevalence.

In proof-of-principle experiments for their PNAS paper, for example, the researchers found that they could detect 14 of 17 verified HGSOC cases based on the mutation burden in TP53 in the peritoneal fluid samples. The approach also led to two false positives in the unaffected control group, corresponding to a sensitivity of 82 percent and 90 percent specificity.

The study’s authors are interested in continuing to examine the relationship between mutational load and cancer development in HGSOC and in other ovarian cancer types, including studies on sample types that are more easily obtained such as Pap smears. They are also interested in taking a closer look at the low-frequency mutations that occur the peripheral blood of healthy individuals as they age.

“There is a general interest in how these mutations accumulate with age in normal people,” Risques said. “That will be very interesting to explore in tissues such as blood and see … the [mutational] load, if it’s different in different genes, how it accumulates with age, and then if it’s a risk factor for cancer, in general.”

To read this entire article on GenomeWeb.com, click here.

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