IUsing 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 Jan. 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, 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.
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.
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.
The cost of the test could be similar to current
cervical fluid HPV testing, which is less than $100,
says graduate student Yuxuan Wang.
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.
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.
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