Glimmer of Hope: Johns Hopkins Uses Pap Smear Test Cervical Fluid to Detect Ovarian & Endometrial Cancers

Using cervical fluid obtained during routine Pap tests, scientists at the Johns Hopkins Kimmel Cancer Center have developed a test to detect ovarian and endometrial cancers. The investigators note that larger-scale studies are needed prior to clinical use on women. 

Using cervical fluid obtained during routine Pap tests, scientists at the Johns Hopkins Kimmel Cancer Center have developed a test to detect ovarian and endometrial cancers. Results of the experiments are published in the January 9 issue of the journal Science Translational Medicine.

In a pilot study, the “PapGene” test, which relies on genomic sequencing of cancer-specific mutations, accurately detected all 24 (100 percent) endometrial cancers and nine of 22 (41 percent) ovarian cancers. The endometrial cancers may have been easier to find because cells from those tumors do not have as far to travel as ovarian cancer cells. The Hopkins researchers will study whether inserting the Pap brush deeper, testing during different times of the menstrual cycle, or other factors might improve detection of ovarian cancer.

The investigators note that larger-scale studies are needed prior to clinical use on women, but they believe the test has the potential to pioneer genomic-based, cancer screening tests. [Emphasis added]

The Papanicolaou (Pap) test, during which cells collected from the cervix are examined for microscopic signs of cancer, is widely and successfully used to screen for cervical cancers. Today, many women’s Paps undergo an additional DNA-based test to see if they harbor the human papillomavirus (HPV), which can spur cervical cancer. However, no routine screening method is available for ovarian or endometrial cancers.

 Luis Diaz, M.D.

Luis Alberto Diaz, M.D.

Since the Pap test occasionally contains cells shed from the ovaries or endometrium, cancer cells arising from these organs could be present in the fluid as well, says Luis Diaz, M.D., associate professor of oncology at Johns Hopkins, as well as director of translational medicine at the Ludwig Center for Cancer Genetics and Therapeutics and director of the Swim Across America Laboratory, also at Johns Hopkins. The laboratory is sponsored by a volunteer organization that raises funds for cancer research through swim events. “Our genomic sequencing approach may offer the potential to detect these cancer cells in a scalable and cost-effective way,” adds Diaz.

Hear Dr. Diaz discuss the PapGene test research in this hyperlinked podcast, courtesy of the American Association for the Advancement of Science.

Cervical fluid of patients with gynecologic cancer carries normal cellular DNA mixed together with DNA from cancer cells, according to the investigators. The investigators’ task was to use genomic sequencing to distinguish cancerous from normal DNA.

The scientists had to determine the most common genetic changes in ovarian and endometrial cancers in order to prioritize which genomic regions to include in their test. They searched publicly available genome-wide studies of ovarian cancer, including those done by other Johns Hopkins investigators, to find mutations specific to ovarian cancer. Such genome-wide studies were not available for the most common type of endometrial cancer, so they conducted genome-wide sequencing studies on 22 of these endometrial cancers.

From the ovarian and endometrial cancer genome data, the Johns Hopkins-led team identified 12 of the most frequently mutated genes in both cancers and developed the PapGene test with this insight in mind.

The investigators then applied PapGene on Pap test samples from ovarian and endometrial cancer patients at The Johns Hopkins Hospital, Memorial Sloan-Kettering Cancer Center, the University of São Paulo in Brazil and ILSbio, a tissue bank. The new test detected both early- and late-stage disease in the endometrial and ovarian cancers tested. No healthy women in the control group were misclassified as having cancer.

Animation of PapGene:

Looking ahead, the investigators’ next steps include applying PapGene on more samples and working to increase the test’s sensitivity in detecting ovarian cancer. “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,” says Chetan Bettegowda, M.D., Ph.D., assistant professor of neurosurgery at Johns Hopkins and a member of the Ludwig Center as well.

Together, ovarian and endometrial cancers are diagnosed in nearly 70,000 women in the United States each year, and about one-third of them will die from it. “Genomic-based tests could help detect ovarian and endometrial cancers early enough to cure more of them,” says graduate student Yuxuan Wang, who notes that the cost of the test could be similar to current cervical fluid HPV testing, which is less than $100.

PapGene is a high-sensitivity approach for the detection of cancer-specific DNA mutations, according to the investigators; however, false mutations can be erroneously created during the many steps — including amplification, sequencing and analysis — required to prepare the DNA collected from a Pap test specimen for sequencing. This required the investigators to build a safeguard into PapGene’s sequencing method, designed to weed out artifacts that could lead to misleading test results.

“If unaccounted for, artifacts could lead to a false positive test result and incorrectly indicate that a healthy person has cancer,” says graduate student Isaac Kinde.

Kinde added a unique genetic barcode — a random set of 14 DNA base pairs — to each DNA fragment at an initial stage of the sample preparation process. Although each DNA fragment is copied many times before eventually being sequenced, all of the newly copied DNA can be traced back to one original DNA molecule through their genetic barcodes. If the copies originating from the same DNA molecule do not all contain the same mutation, then an artifact is suspected and the mutation is disregarded. However, bonafide mutations, which exist in the sample before the initial barcoding step, will be present in all of the copies originating from the original DNA molecule.

The Johns Hopkins test results demonstrate that DNA from most endometrial and a fraction of ovarian cancers can be detected in a standard liquid-based Pap smear specimen obtained during routine pelvic examination. Although improvements need to be made before applying this test in a routine clinical manner, it represents a promising first step toward a broadly applicable screening methodology for the early detection of gynecologic malignancies.

“This is very encouraging, and it shows great potential,” said American Cancer Society genetics expert Michael Melner.

“We are a long way from being able to see any impact on our patients,” cautioned Dr. Shannon N. Westin of the University of Texas MD Anderson Cancer Center. Dr. Westin reviewed the research in an accompanying editorial, and said the ovarian cancer detection would need improvement if the test is to work. But Dr. Westin noted that ovarian cancer has poor survival rates because it’s rarely caught early. “If this screening test could identify ovarian cancer at an early stage, there would be a profound impact on patient outcomes and mortality,” Westin said.

More than 22,000 U.S. women are diagnosed with ovarian cancer each year, and more than 15,000 die. Symptoms such as bloating and pelvic or abdominal pain are seldom obvious until the cancer is more advanced, and numerous attempts at screening tests have failed.

Endometrial cancer affects about 47,000 U.S. women a year, and kills about 8,000. There is no screening test for it either, but most women are diagnosed early because of postmenopausal bleeding.

___________________________

Funding for the research was provided by Swim Across America, the Commonwealth Fund, the Hilton-Ludwig Cancer Prevention Initiative, the Virginia & D.K. Ludwig Fund for Cancer Research, the Experimental Therapeutics Center of the Memorial Sloan-Kettering Cancer Center, the Chia Family Foundation, The Honorable Tina Brozman Foundation, the United Negro College Fund/Merck Graduate Science Research Dissertation Fellowship, the Burroughs Wellcome Career Award for Medical Scientists, the National Colorectal Cancer Research Alliance and the National Institutes of Health’s National Cancer Institute (N01-CN-43309, CA129825, CA43460).

In addition to Kinde, Bettegowda, Wang and Diaz, investigators participating in the research include Jian Wu, Nishant Agrawal, Ie-Ming Shih, Robert Kurman, Robert Giuntoli, Richard Roden and James R. Eshleman from Johns Hopkins; Nickolas Papadopoulos, Kenneth Kinzler and Bert Vogelstein from the Ludwig Center at Johns Hopkins; Fanny Dao and Douglas A. Levine from Memorial Sloan-Kettering Cancer Center; and Jesus Paula Carvalho and Suely Kazue Nagahashi Marie from the University of São Paulo.

Papadopoulos, Kinzler, Vogelstein and Diaz are co-founders of Inostics and Personal Genome Diagnostics. They own stocks in the companies and are members of their Scientific Advisory Boards. Inostics and Personal Genome Diagnostics have licensed several patent applications from Johns Hopkins. These relationships are subject to certain restrictions under The Johns Hopkins University policy, and the terms of these arrangements are managed by the university in accordance with its conflict-of-interest policies.

____________________________

Citations:

I. Kinde, C. Bettegowda, Y. Wang, J. et. al. Evaluation of DNA from the Papanicolaou Test to Detect Ovarian and Endometrial Cancers. Sci. Transl. Med. 5, 167ra4 (2013).

S. N. Westin, G. B. Mills, A. P. Myers, Repurposing the Pap Smear: One Step Closer to Gynecologic Cancer Screening. Sci. Transl. Med. 5, 167ps1 (2013).

Additional Sources:

Johns Hopkins Scients Use Pap Test Fluid to Detect Ovarian, Endometrial Cancers, John Hopkins Medicine, Press Release, January 9, 2013.

Retooling Pap Test To Spot More Kinds Of Cancer, The Associated Press via National Public Radio, January 9, 2013.

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