URINE TEST TO DETECT CERVICAL CANCER

JHU specialists reported they have developed a urine test for the likely emergence of cervical cancer that is highly accurate compared to other tests based on genetic markers derived directly from cervical tissue. The new urine test, they said, is different because it analyzes not only multiple sources of human cellular DNA altered by precancerous changes, but also DNA from human papillomavirus (HPV) that is sexually transmitted and causes virtually all cases of the disease.

In a proof-of-concept study, described by Guerrero-Preston et al in Cancer Prevention Research, the investigators said their genetic markers test showed a sensitivity of 90.9% in identifying so-called cervical intraepithelial neoplasia 2 lesions. Additionally, they demonstrated that the DNA for all three human genes and one viral gene could be successfully extracted from urine, and they could identify such lesions with 75% sensitivity.

Two commercial tests based on markers of DNA chemical changes called methylation released in Europe last summer require Pap smears or swabs of cervical tissue, and show 64% sensitivity in identifying similar lesions, according to senior investigator Rafael Guerrero-Preston, DrPH, MPH, Assistant Professor of Otolaryngology–Head and Neck Surgery at the Johns Hopkins University School of Medicine and member of the Johns Hopkins Kimmel Cancer Center.

“If further studies confirm these findings, we see a significant use of urine screening as a way to quickly and inexpensively determine if a biopsy is warranted, or if physicians can use a ‘watch and wait’ approach before intervening,” said Dr. Guerrero-Preston. Typically, he said, a woman who tests positive for HPV and has an abnormal Pap smear undergoes a biopsy to rule out cervical cancer using cells taken directly from cervical tissue. But previous studies suggest more than 50% of these biopsies are unnecessary and can result in pain, worry, infertility, and higher health-care costs.

“Our urine test would serve as a molecular triage,” he said, “at times supplementing Pap test information. In developing countries that don't have the money, medical infrastructure, or cultural approval for Pap test, our test could be used instead.”

Study Details

The new study builds on the Johns Hopkins team's previously published work, in which investigators identified three genes associated with cervical cancer or abnormal-looking cells known to become cancerous: FKBP6, INTS1, and ZNF516. As abnormalities progress, these genes were more likely to have a chemical methyl group attached to their DNA in certain spots.

The researchers tested the value of these genes as markers using 214 cervical cell samples collected from women undergoing Pap smears at Hospital Dr. Hernan Henriquez Aravena in Chile. The women ranged in age from 18 to 86. Among the test samples, 34 showed no abnormalities in their cervix; 87 showed 1 of 3 types of precancerous, abnormal tissue; and 90 showed clear evidence of cervical cancer.

Next, Dr. Guerrero-Preston's team isolated DNA from each cervical tissue sample and used advanced genetic sequencing methods to spell out the DNA makeup of cells in the cervical tissue samples. The researchers then compared the number of methyl groups attached to each gene in samples from the 34 healthy women to 53 samples with a specific subset of precancerous markers.

Sensitivity and Specificity

Using methylation as a value to diagnose malignancy, the three genes together showed a 90% sensitivity and an 88.9% specificity. To further improve the accuracy of the test, the investigators added a new gene marker to the test. This time, rather than using a human gene, they used one from the virus, HPV16-L1, which also becomes methylated in human cells as cancer develops.

They conducted the test again with the four-gene combination on a new population of women from the University of Puerto Rico. The 115 women ranged in age from 21 to 49; 41 participants had healthy cervical tissue, and 74 had one of three types of precancerous cells. Using all four genes, the test now had a sensitivity of 90.9% and a specificity of 60.9%.

“When developing a new cancer screening test, we want something in the range of 90% to 95% sensitivity, which is competitive with the effectiveness of tests developed and now marketed in Europe,” said Dr. Guerrero-Preston.

The next step, the researchers reported, was to verify that the four-gene test worked using freely circulating DNA from blood and urine, rather than DNA taken directly from cervical tissue. For this experiment, they tested 40 samples of paired cervical tissue, blood, and urine from a subset of the patients from Puerto Rico. Using the DNA from blood, they found the test had an 85.7% sensitivity and a 60.9% specificity. Using urine, they found a 75% sensitivity and an 83.3% specificity.

During the course of the study, the time to process cervical samples, blood, or urine and get a test result took 4 days. They plan to continue modifying the test to improve the urine sensitivity to that of the cervical tissue samples.