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. 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. Results of the experiments are published in the January 9 issue of the journal, Science Translational Medicine.
The investigators note that larger scale studies are needed before clinical implementation can begin, but they believe the test has the potential to pioneer genomic-based cancer screening tests.
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. However, no routine screening method is available for ovarian or endometrial cancers.
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 and director of the Swim Across America Laboratory. The Laboratory is sponsored by a volunteer organization that raises funds for cancer research. “Our genomic sequencing approach may offer the potential to detect these cancer cells in a scalable and cost effective way,” adds Diaz.
Cervical fluid of patients with gynecologic cancer carries normal cellular DNA mixed together with DNA from cancer cells, according to the investigators. Their 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 publically-available genome-wide studies of ovarian cancer, including those done by other Johns Hopkins investigators, to find ovarian-cancer specific mutations. 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 Sao 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.
Early detection of ovarian cancer saves women’s lives. No screening test exists that can test all women for ovarian cancer. The Pap test does not test for ovarian cancer; it screens for cervical cancer.
Not only do researchers need to develop an early detection test for ovarian cancer, like mammograms for breast cancer and Pap tests for cervical cancer, but also women and medical professionals need to become more aware of ovarian cancer symptoms.
While no early detection tool exists for all women, several tests exist for women who are at a high risk. If a woman has ovarian cancer symptoms, a strong family history, or a genetic predisposition such as a BRCA mutation, doctors may monitor her with one of three tests or a combination of them:
The protein CA-125 exists in greater concentration in cancerous cells. Though a high count of this protein may help doctors identify ovarian cancer, premenopausal women may have an elevated CA-125 due to benign conditions unrelated to ovarian cancer. Uterine fibroids, liver disease, inflammation of the fallopian tubes, and other types of cancer can raise a woman’s CA-125 level, often causing a false positive test for ovarian cancer.
Although the CA-125 blood test is more accurate in postmenopausal women, it is not a reliable early detection test for ovarian cancer. In about 20 percent of advanced stage ovarian cancer cases and 50 percent of early stage cases, the CA-125 is not elevated even though ovarian cancer is present. As a result, doctors generally use the CA-125 blood test in combination with a transvaginal ultrasound.
The CA-125 blood test can be an important tool for evaluating the disease’s progress and tumors’ response to treatment. Additionally, this test can monitor a woman’s CA-125 level for evidence of recurrence.
OVA1 has also been approved by the Food and Drug Administration (FDA) for risk stratification. A woman who presents with a known tumor may have this test to determine if her surgery should be done by a gynecologist or a gynecologic oncologist – doctors who are specially trained to treat women with gynecologic cancers.
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.
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.
A transvaginal ultrasound is a test used to examine a woman’s reproductive organs and bladder.
To administer the test, the doctor inserts a probe into the woman’s vagina. The probe sends off sound waves which reflect off body structures. The waves are then received by a computer that turns them into a picture.
A pelvic exam should be a part of a woman’s regular female health exam.
This exam requires the doctor to place one or two fingers into a woman’s vagina and another over her abdomen to feel the size, shape, and position of the ovaries and uterus. Ovarian cancer is rarely detected in a pelvic exam and usually in an advanced stage if it is.
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. The investigators needed 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.
Researchers have been hoping to find a screening test that would help detect ovarian cancer early, much like prostate-specific antigen, or PSA, tests do with prostate cancer and mammograms do for breast cancer. But the best tests to date - a CA 125 blood test and a transvaginal sonogram - give many false positives as well, so neither are currently recommended for screening the population at large.
Both tests are recommended for women who have a family history of ovarian cancer, or who experience symptoms such as abdominal bloating, pain, change in bowel habits or unusual fatigue. It’s unclear how big a tumor has to be before such symptoms are evident, but even when they are, women often ignore them and doctors often dismiss them as gastrointestinal upset.
In a recent survey by the National Ovarian Cancer Coalition, a nonprofit advocacy group, 83% of women said their doctors had not discussed the symptoms of ovarian cancer with them, and 62% of Americans believe - erroneously - that a Pap smear can diagnose it.
“Nobody knows your body as well as you do. If the symptoms are there, pay attention,” says April Donahue, an ovarian-cancer survivor from Quakertown, Pa. When she was 24, she went to four different doctors for pain, fatigue and bloating before one removed what he thought was an ovarian cyst. It turned out to be cancer. Ten years later, Ms. Donahue’s symptoms returned, and doctors again brushed it off. But she insisted on having her remaining ovary removed, and cancer was discovered again. “Hey, I’m lucky. I’m an 18-year survivor, and not a lot of women can say that,” she says.
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.
Funding for the research was provided by Swim Across America, the Commonwealth Fund, the Hilton-Ludwig Cancer Prevention Initiative, the Virgina and D.K. Ludwig Fund for Cancer Research, the Experimental Therapeutics Center of Memorial Sloan-Kettering Cancer Center, the Chia Family Foundation, The Honorable Tina Brozman Foundation, The United Negro College Fund-Merck Graduate Science 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 Center (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, 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; Jesus Paula Carvalho and Suely Kazue Nagahashi Marie from the University of Sao 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.
Johns Hopkins Medicine