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For HPV-Positive Women, Test Can Guide Cervical Cancer Screening Follow-Up

, by NCI Staff

Dual-stain testing on Pap test samples identifies the presence of two proteins: p16 (brown) and Ki-67 (red).

Credit: National Cancer Institute

UPDATE: On March 11, 2020, the Food and Drug Administration (FDA) approved the first dual-stain test for women who have tested positive for HPV. The test, called CINtec® PLUS Cytology, is used to help doctors decide if an HPV-positive woman should have a biopsy to look for cervical precancer or cancer.

A new test, called dual stain, may help improve how doctors care for women who test positive for human papillomavirus (HPV) infection during routine cervical cancer screening, an NCI-led study has shown. HPV tests detect infections with the cancer-causing types of HPV.

For most women, HPV infections go away on their own. But for some, the infection leads to precancerous growths that can progress to cervical cancer. To better care for women who are HPV positive, researchers have been exploring ways to help tell whether an HPV infection is likely to cause precancer.

As HPV testing becomes a more common method of cervical cancer screening, “the challenge is how to best manage, or triage, HPV-positive women,” said the study’s senior investigator, Nicolas Wentzensen, M.D., Ph.D., of NCI’s Division of Cancer Epidemiology and Genetics (DCEG).

Doctors usually use a Pap test, also called a Pap smear, to determine whether an HPV infection is likely to cause precancer and help decide whether a woman should get a biopsy. But, according to the NCI study, the dual-stain test was better than the Pap test at predicting whether HPV-positive women developed cervical precancer within 5 years.

The findings suggest that HPV-positive women with a positive dual-stain test result should get a biopsy to check for cervical precancer or cancer, the study authors concluded, whereas those with a negative result can safely wait 3 years before getting screened again. Results from the prospective study were reported October 11 in JAMA Oncology.

“This is a very important study,” said Mark Stoler, M.D., associate director of Surgical Pathology and Cytopathology at the University of Virginia School of Medicine. It’s the first study performed in the United States, Dr. Stoler continued, “that really provides the assurance that [dual stain] is a better triage test” than the Pap test.

Cervical Cancer Screening in the United States

There are around 12 different types of HPV that can cause cervical and other types of cancer. HPV infections that aren’t controlled by the immune system are the cause of nearly all cervical cancers. Cervical cancer screening tests look for disease in people who have no symptoms.

Current US guidelines for cervical cancer screening recommend one of three approaches: an HPV test alone, a combination of HPV and Pap tests (an HPV/Pap cotest), or a Pap test alone.

According to the US Preventive Services Task Force, women age 30 to 65 years at average risk of cervical cancer can be safely screened with an HPV test or HPV/Pap cotest every 5 years.

Although HPV/Pap cotesting has improved the accuracy of cervical cancer screening and helped to greatly reduce the incidence of cervical cancer, it has some limitations. 

For example, some women who test positive for HPV and who have minor abnormalities on a Pap test are referred for colposcopy, a procedure in which biopsies are taken of abnormal areas in the cervix (visit NCI's page on cervical screening tests for the most up-to-date recommendations). 

But only a small percentage of Pap test abnormalities turn out to be cervical precancer or cancer, meaning most of these women had an unnecessary colposcopy, Dr. Wentzensen explained. Because of these limitations, “there is a big effort to find better markers that allow us to triage HPV-positive women more efficiently,” he said.

In terms of potential triage approaches for HPV-positive women, dual-stain testing “is probably the most advanced method available,” said the study’s lead investigator, Megan Clarke, Ph.D., M.H.S., also of DCEG. The new study adds to that body of evidence, she said.

The dual-stain approach has been under study for more than a decade, Dr. Stoler said. It measures the presence of two specific proteins, p16 and Ki-67, in a sample of cervical cells. The expression of p16 is strongly linked with HPV infection, and Ki-67 is used as a biomarker for the rapid cell division seen in precancers and cancer. 

Previous studies have provided some evidence that dual-stain testing is better at identifying HPV-positive women who have precancers than Pap testing, but none followed patients forward in time for more than 3 years.

Searching for a Better Triage Test for HPV-Positive Women

To conduct this new study, the investigators followed 1,549 women aged 30 or older who had tested positive for HPV (on a test that gives combined results for 13 cancer-causing types) while undergoing routine HPV/Pap cotesting at Kaiser Permanente Northern California between January and May 2012. Dual-stain testing was performed on participants’ cervical cell samples at the beginning of the study.

Women with a normal Pap test result were advised to repeat the cotest in 1 year, whereas women with an abnormal result were referred for immediate colposcopy and biopsy. Biopsy results can reveal a range of disease states, including normal, low-grade to high-grade precancer, and cancer.

Overall, 46% of women in the study had a positive dual-stain test result and 51% had an abnormal Pap test result. More women with severely abnormal Pap test results than women with normal Pap results had a positive dual-stain test result. Over the 5-year study period, 77% of women found to have a high-grade precancer and 91% found to have cancer had a positive dual-stain test result.

Compared with Pap test results, dual-stain test results were far more indicative of the 5-year risk of cervical precancer, the team found. For example, women with a positive dual-stain test result had a higher risk of developing cervical precancer over the next 5 years than women with a positive Pap test result. 

Conversely, women with a negative dual-stain test result had a lower risk of developing cervical precancer within 5 years, compared with women with a normal Pap test result. 

This means that a negative dual-stain test result gives greater reassurance than a normal Pap test result that precancer won’t develop during the ensuing 5 years, Dr. Wentzensen explained.

Dr. Clarke said that the study also addressed a critical question: “How often and at what time interval should HPV-positive women who test negative with dual stain come back for repeat screening?”

Based on the 5-year risks of cervical precancer, the researchers determined that HPV-positive women with a negative dual-stain result can safely wait 3 years before being screened again. 
Together, the findings suggest that using the dual-stain test to triage HPV-positive women might lead to fewer unnecessary colposcopies, Dr. Clarke noted.

The higher sensitivity (i.e., better at identifying women with a higher risk of having precancer) and specificity (i.e., better at identifying those with a low risk) of the dual stain-test compared with the Pap test is impressive, Dr. Stoler said.

“It’s very hard to have a test that improves sensitivity and specificity, but dual stain does it because of the biology behind the development of the test,” he explained.

Learning More About Dual Stain

The dual-stain test is already being marketed and used in several countries, including Canada, Europe, and Australia, Dr. Stoler said. The clinical trial that would form the basis of FDA clearance for the dual-stain test in the United States is currently ongoing, he added.

In the future, HPV testing followed by dual stain for primary cervical cancer screening may be a more efficient alternative to HPV/Pap cotesting, Drs. Wentzensen and Clarke said.

The manufacturer of the dual-stain test is analyzing the development of cervical precancer in HPV-positive women who have a positive test result from either dual stain or Pap in an ongoing study. In addition, Dr. Wentzensen and his colleagues are investigating the results of dual stain testing in a large population of women, including many who are HPV negative.

“I think there will be sufficient data to change practice based on these observational studies,” said Dr. Wentzensen.

And the test may soon have other attractive qualities, the investigators noted. A major advantage of the dual-stain test, said Dr. Wentzensen, is that abnormal cells are highlighted with a colored stain and are therefore easier to detect and quantify than cells stained for a Pap test.

“Currently, the dual-stain test is evaluated manually, but we are working on an automated evaluation of this assay and we have exciting results,” he added. Automation of the dual-stain test would enhance its reliability and make it easier to use, he said.