Skip to main content
Premium Trial:

Request an Annual Quote

Turning Pap Test into Sequencing-Based Gynecologic Cancer Assay will Take Further Validation


Following last month's publication of a study that suggested the Papanicolaou test, or Pap smear, could potentially be repurposed as a sequencing-based screening test for endometrial and ovarian cancer, the authors are now working on validating the results, hoping to develop a routine clinical test for the early detection of cancer.

The initial results, described in a proof-of-concept study published in Science Translational Medicine last month, were promising. Using DNA collected in routine Pap smears, the test detected all cases of endometrial cancer and some of ovarian cancer, and produced no false positive results.

"Our next step is to just reproduce this in hundreds of samples rather than in scores of samples, and that's ongoing now," said Luis Diaz, an associate professor of oncology at Johns Hopkins University and the senior author of the study. He said those follow-up studies would also involve cervical cancers.

Diaz said he and his colleagues have been working on early cancer detection for many years, focusing on circulating tumor DNA and detecting rare mutations in complex biological fluids, such as blood.

When talking to a group of obstetricians and gynecologists, he said, the idea came up that ovarian and endometrial cancer cells might travel into the uterus and some might collect in the cervix, where they could be picked up in a routine Pap smear sample, which is taken with a brush from the endocervical canal, and identified by unique mutations in their DNA.

While a blood test for cancer "would be the best thing in the world," Diaz said, using Pap smear samples appeared promising because DNA from gynecologic cancers would likely be enriched in the cervix, and it would be easier to trace any cancer mutations found back to the organ where the cancer cells originated.

For their study, the researchers initially sequenced either the exomes or a targeted set of frequently mutated genes in the DNA of 46 tumors, 24 from endometrial and 22 from ovarian cancer patients, and they were able to find at least one somatic mutation in each.

Next, they sequenced genes that they knew were mutated in these cancer samples in
DNA from liquid-based Pap smear samples from the same 46 patients, which were obtained from the endocervical canal using a brush.

Because the majority of the DNA in the samples was expected to come from normal cervical cells, and only a small fraction from cancer cells, the researchers analyzed the DNA using a previously published technique called Safe-Sequencing System, or Safe-SeqS (CSN 6/1/2011), which is designed to distinguish in the sequencing reads between bona fide mutations and errors introduced during PCR amplification or the sequencing process.

For the endometrial cancer patients, the researchers were able to find a mutation present in the cancer in every single Pap smear specimen, while for the ovarian cancer patients, they found mutations in 9 of the 22, or 41 percent, of the Pap specimens.

Subsequently, the researchers designed a Safe-SeqS prototype screening assay, which they called "PapGene," that assesses 50 amplicons from a total of 12 genes most commonly mutated in endometrial and ovarian cancer. In preliminary experiments, they showed that 46 of these amplicons were able to detect mutations contained in at least 0.1 percent of template molecules.

The test runs on the Illumina sequencing platform. The researchers have been using both the HiSeq and MiSeq for their analyses, Diaz said, depending on the number of samples they need to analyze.

They applied the PapGene test to Pap specimens from 12 of the endometrial and two of the ovarian cancer patients, as well as 14 control samples, and were able to detect all mutations expected to be present in the cancer samples, while not detecting any mutations in the controls.

"The most important finding in this paper is that diagnostically useful amounts of cells or cell fragments from endometrial and ovarian cancers are present in the cervix and can be detected through molecular genetic approaches," the authors noted in their paper.

Right now, Diaz and his colleagues are attempting to replicate these results in a larger number of samples. Along the way, they also plan to make improvements to the assay.

One bottleneck is obtaining enough samples from ovarian cancer patients that also have Pap smear specimens available, he said, but the hope is that the follow-up study will be completed in about a year.

The researchers also want to expand the panel of genes, or regions within those genes, in the test. Right now, that number is limited by the sensitivity of the test, but "as the technology of Safe-SeqS advances, we think that we will be able to do many, many more," Diaz said.

Improving the technical sensitivity of the PapGene test would also mean it could potentially pick up more ovarian cancers.

Another idea is to improve the Pap test collection method to obtain a greater number of cells from the endometrium and ovaries, for example by having the brush reach into the transition zone of the cervix or, later, by using an instrument that reaches up into the endometrial cavity. "If you simply assess for that quality [of the Pap smear], we can potentially improve the yield [of the test]," Diaz said.

If the follow-up study confirms the initial results, the researchers plan to perform a blinded prospective study, involving women predisposed to ovarian cancer as well as the general population. Depending on the test's sensitivity, such a study would involve in the high hundreds to low thousands of women, Diaz said.

Peter Bach, a physician and cancer epidemiologist at Memorial Sloan-Kettering Cancer Center, agreed that a broad-based validation of the proof-of-concept results will be crucial, followed by studies showing the results are reproducible across populations. "And then there is a very important issue, whether or not screening matters — how it affects mortality," he said.

"We've learned the hard way that you can't skip over that step because even in places where a test would help us find many early cancers, it's frequently surprising how ineffective it is at reducing burden from the disease," he said.

"Certainly I would not want to bet either way that this would become standard of care or anything like that until we know a lot more about it."

If the results pan out, though, the PapGene test could become a routine screening test for endometrial and ovarian cancer, both for women with an increased family or genetic risk for the diseases and for older women, Diaz said.

The cost of the PapGene test today is about the same as that of the human papillomavirus test — about $100 — which is routinely conducted as part of the Pap smear, Diaz said. According to the authors, that cost is expected to drop further with decreased sequencing costs.

Piggy-backing on an existing test seems like a good idea, according to Bach, given current healthcare costs. "We've already got a pretty long list of things we want most individuals to do for disease prevention and screening, so this is very appealing," he said.

He questioned, though, whether the frequency of Pap smears — most women with normal results do not get them every year — would match the frequency needed for an endometrial and ovarian cancer screening test.

Diaz said he and his colleagues have talked to several companies interested in developing the test commercially and "would be interested in talking to any commercial partners that wanted to evolve this technology."

According to the paper, two companies founded by Diaz and colleagues — Inostics and Personal Genome Diagnostics — have licensed several patent applications from Johns Hopkins that are relevant to the test, among them WO2012/142,213, “Safe Sequencing System,” and provisional patent application 61/719,942, “Papanicolaou test for ovarian and endometrial cancers.”