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Study Suggests DNA from Pap Test Samples Can Be Used to Detect Ovarian, Endometrial Cancers

NEW YORK (GenomeWeb News) – The same liquid samples collected during many modern-day Papanicolaou, or "Pap," tests for cervical abnormalities and human papillomavirus infection may serve as a resource for finding genetic changes linked to endometrial and ovarian cancers, according to a study online today in Science Translational Medicine.

"Although improvements need to be made before applying this test in a routine clinical manner," the study authors argued, "it represents a promising step toward a broadly applicable screening methodology for the early detection of gynecologic malignancies."

Using existing genetic data on ovarian tumors and newly generated exome sequences for nearly two-dozen endometrial tumors, investigators from Johns Hopkins University and elsewhere in the US and Brazil started by sussing out which genes are most frequently mutated in the gynecological cancers.

From there, the team went on to show that mutations found in ovarian or endometrial tumors often tended to turn up in sequences from matching Pap test fluid samples, particularly in individuals with endometrial cancer.

And by bringing such findings together, the investigators narrowed in on a panel of oft-mutated genes, which dubbed "PapGene," that appears to show promise for finding endometrial or ovarian cancer-related mutations in fluid collected during Pap tests.

Though more research is needed to assess the potential genetic test and its utility in the clinic, they said, results so far hint that sequencing the PapGene panel may broaden the information that can be obtained from the sorts of patient samples already being collected routinely in the clinic.

"Our genomic sequencing approach may offer the potential to detect these cancer cells in a scalable and cost-effective way," co-senior author Luis Diaz, Jr., a researcher with the Ludwig Center for Cancer Genetics and Therapeutics at Johns Hopkins University and director of the institute's Swim Across America Laboratory, said in a statement.

Pap tests involve the collection of cervical cells to look for signs of cervical cancer or pre-cancerous lesions. For liquid-based versions of the test, which are often used today, study authors said, clinicians preserve some of the sample in a liquid form. That sample can then be used to test for the presence of DNA from HPV, a virus that sometimes leads to cervical cancer.

But Diaz and his colleagues suspected that cervical fluid might contain other informative genetic material as well. In particular, they speculated that cancers occurring in nearby endometrial or ovarian tissues might slough off mutation-containing DNA that would make its way to the cervix and get caught up in the Pap sample.

To begin testing that theory, the researchers started by trying to take stock of the most common mutations in some gynecological cancers.

Along with analyses of existing ovarian and endometrial tumor data, the team performed exome sequencing on matched tumor and normal samples from 22 individuals with endometrioid cancer, the most common endometrial cancer subtype.

After capturing protein-coding regions in the tumor and normal samples with an Agilent SureSelect Human Exome kit, the team used Illumina's GAIIx instrument to sequence each of the exomes to an average depth of around 149-fold over more than 88 percent of the bases targeted.

Within the 22 tumors tested, mutation profiles clustered in two broad groups, the researchers noted. A dozen tumors harbored more than 100 predicted somatic coding mutations (perhaps owing to alterations affecting DNA repair pathways), while the remaining 10 tumors had fewer than 100 somatic mutations apiece.

By folding in existing data on ovarian cancers and endometrial tumors from other subtypes, the investigators came up with a list of the top mutation-containing candidate genes across the gynecological cancer types.

Analyses of targeted or whole-genome sequence data on another 24 endometrial tumor samples and 22 ovarian tumor samples verified the notion that the team's gene list contained authentic cancer players: Each of the 46 tumors tested harbored a mutation within at least one of the genes.

And that prompted researchers to go a step further, testing for DNA alterations in Pap test fluid samples collected from the same 46 women whose tumors had been tested directly. There, researchers found tumor-related mutations in cervical fluid samples from all 24 women with endometrial cancer and in nine of the 22 women with ovarian cancer.

Similarly, investigators found mutations in genes from their PapGene panel through targeted sequencing on Pap test samples from two more women with ovarian cancer and another 12 women with endometrial cancer. In contrast, such alterations did not turn up in Pap test samples from 14 unaffected women.

Moreover, the sequencing-based method uncovered mutations in the PapGene panel in individuals with both early stage cancers and those whose cancers had progressed to later stages.

In an effort to ward off false-positive PapGene tests caused by amplification, sequencing, or other errors, the researchers used the so-called "Safe-SeqS" method to sequence the targeted genes in each of the cervical fluid samples.

That involves slapping 14-base DNA barcodes onto DNA fragments in samples prior to amplification and analyses so that the team can check back to see if apparent mutations in the cancer genes are present in all amplicons from the same stretch of sequence.

Going forward, the group hopes to make additional improvements to the PapGene test and to further explore its utility.

"Performing the test at different times during the menstrual cycle, inserting the cervical brush deeper into the cervical canal, and assessing more regions of the genome may boost the sensitivity," said JHU neurosurgery researcher Chetan Bettegowda, one of the first authors on the study, in a statement.

In an accompanying perspectives article in the same issue of Science Translational Medicine, researchers from the University of Texas' MD Anderson Cancer Center and the Dana-Farber Cancer Institute discussed the PapGene sequencing strategy within the context of past and current schemes for diagnosing such gynecological diseases.

They also touched on some of the issues that would need to be addressed before the test enters the clinic and noted that a similar approach may eventually offer clues about the biological processes involved in the development of such cancers.

For instance, along with the sensitivity and specificity of the genetic test, they said that it will likely be important to consider its ease and affordability. They also noted that the advent of screening strategies for ovarian and endometrial cancers based on Pap test samples may prompt changes to the frequency with which the tests are administered and/or the age of the patient groups targeted.