Dual-stain cytology is evolving cervical-cancer diagnostics

Discover the advancements in cervical-cancer diagnosis and its future.

July 10, 2023

Ancestral forms of one type of human papillomavirus (HPV) have most likely infected all human populations since Homo erectus.¹Today, 80% of people in the U.S. get HPV by age 45, with 13,290 cases expected to manifest into cervical cancer in 2023.²

Fortunately for women in the 20th century, the diagnostic Pap smear test was created, which helped greatly reduce these numbers. The Pap smear can detect potentially precancerous and cancerous processes in the cervix.

While Pap cytology has served women well over the decades, technology has evolved. In the last decade, advances have been made in HPV testing that allow OB-GYNs to know whether their patients have an HPV genotype that places them at higher risk of cervical cancer.

More recently, a new technology called dual stain has been available to determine if a patient’s HPV is transforming into pre-cancer. When detected at an early stage, the five-year survival rate for women with invasive cervical cancer is 92%.³ The only commercially available dual-stain product is the CINtec PLUS® Cytology dual-stain biomarker triage test approved by the Food and Drug Administration (FDA) in 2020.

Rebecca Khan, M.D., a practicing OB-GYN, who has helped implement dual stain in a large reference laboratory that processed 1.5 million Pap cytology tests annually, provided some answers to questions about dual-stain triage.

What is dual-stain triage?

Dual stain identifies abnormal cells through biomarker staining of cervical cells collected during routine cervical cancer screening. It uses two biomarkers – p16 and Ki-67 – that, when found together, signal changes at the cellular level that indicate a transforming HPV infection.

The biomarkers provide an objective interpretation of the drivers behind transforming HPV infection, the co-expression of halting and progressive cell division. This provides greater risk and disease stratification when screening for cervical cancer.

How does the science behind dual-stain technology work?

p16 is a biomarker that occurs in normal cells and signifies cell cycle arrest. If you think of it like a stoplight, it tells you to stop. It’s indicated by brown staining in the cytoplasm.

Ki-67 is a biomarker that occurs in normal cells and signifies cell division. It’s indicated by the red stain in the nucleus. When the two biomarkers – p16 and Ki-67 – are both in the cell at the same time, it's like a traffic light where the red and green lights are on at the same time and it's like, Whoa, do I stop or do I go? So you know there is something wrong.

The HPV is in the cell, integrating into the DNA and causing transformation before you actually see it in the morphology of the cell. Cytology is subjective but dual stain is a more objective, biomarker-based assay, and you interpret it completely independent of cytology.

What does this information tell us?

Dual stain tells us what a patient's cells are doing in response to HPV infection, independent of their HPV genotype. It tells us whether or not the high-risk HPV E6/E7 are affecting a patient's tumor suppressor proteins, and it tells us if a patient's cells are undergoing oncogenic transformation.

How does this information impact risk?

Under the current guidelines, most patients with discrepant co-testing repeat in 12 months. We know if we send someone away who has NILM with 12 other HPV, she has a 5.7% chance of having a developing CIN2 or greater. If we do CINtec PLUS and send her away after a negative result, we know she has only a 2.9% chance of developing CIN2 or greater.

Also, if you look at the risk of having a known HPV-positive test for HPV 16 or 18, those are the ones we’re taking to colposcopy; their risk is 12.6%. When you have a normal 12-other HPV positive result with a positive CINtec PLUS, the risk is 12%. Because those risks are pretty equal, it makes sense that those people could benefit from colposcopy rather than coming back the next year.

What does the big picture of a dual-stain test mean?

The ATHENA trial showed us that one in four women who are HPV 16 positive will have cervical disease in three years. If we can find those people before they have major abnormalities on their cytology, we have done them a service because we have found them earlier and have a greater chance of preventing CIN3.

How should dual-stain testing be used?

When patients test positive with the cobas HPV test, that same sample can be used with CINtec PLUS, a biomarker test, to determine if the patient is progressing toward cervical cancer. In primary HPV screening, CINtec PLUS Cytology can be used as a triage test instead of Pap cytology.⁴ In co-testing, CINtec PLUS Cytology can be used as a triage test to resolve seemingly discrepant HPV positive/Pap normal screening results.

Contributor

Rebecca Khan, M.D., a practicing OB/GYN, who has helped implement dual stain in a large reference laboratory processed 1.5 million paps annually, provided some answers to questions OB/GYNs have about dual-stain triage.

Guidelines and Evidence

What do the ASCCP cervical cancer guidelines say about dual-stain technology?

New guidelines shared by the American Society for Colposcopy and Cervical Pathology (ASCCP)⁸ and other members of the Enduring Guidelines for Cervical Cancer Screening and Management Committee now include dual-stain triage testing to enable earlier diagnosis. CINtec PLUS Cytology is the only dual-stain product approved by the FDA.

What does the evidence tell us?

The IMPACT trial was a multi-center prospective trial that enrolled 35,000 women at 32 collection sites across the U.S. It was representative of the routine cervical-cancer screening population, including those from ages 25 to 65, unvaccinated and vaccinated, and representing diverse races and ethnicities.

In the IMPACT clinical trial, co-testing with CINtec PLUS Cytology as a triage detected cervical disease earlier in seven out of 10 women who were already identified as HPV positive. This allows clinicians to treat disease earlier, a critical factor in improving cancer patient outcomes.

Two studies, Kaiser Permanente Northern California and the STRIDES in Mississippi, also provide further evidence for the technology’s clinical utility and effectiveness.

Disclaimers: Paid presenter of Roche Diagnostics. This content is provided for educational and informational purposes only and does not constitute providing medical advice or professional services. The information provided should not be used for diagnosing or treating a health problem or disease, and those seeking personal medical advice should consult with a licensed physician. Always seek the advice of your doctor or another qualified health provider regarding a medical condition.

References

Pimenoff VN, Houldcroft CJ, Rifkin RF, Underdown S. The Role of aDNA in Understanding the Coevolutionary Patterns of Human Sexually Transmitted Infections. Genes. June 25, 2018;9(7).
National Cancer Institute. https://seer.cancer.gov/statfacts/html/cervix.html. Last accessed May 26, 2023.
National Cancer Institute. https://www.cancer.gov/types/cervical/survival. Last accessed May 31, 2023.
Wright Jr TC, Stoler MH, Ranger-Moore J, Fang Q, Volkir P, Safaeian M, Ridder R. Clinical Validation of p16/Ki-67 Dual-Stained Cytology Triage of HPV-Positive Women – Results from the IMPACT Trial. Intl J Cancer 2021; https://doi.org/10.1002/ijc.33812.
CINtec® PLUS Cytology. Package insert. Roche Diagnostics; 2020.
Wentzensen N, Clarke MA, Bremer R, et al. Clinical Evaluation of Human Papillomavirus Screening With p16/Ki-67 dual-stain Triage in a Large Organized Cervical Cancer Screening Program. JAMA Intern Med. 2019;179(7):881-888. doi:10.1001/jamainternmed.2019.0306.
Risley C, Stewart MW, Geisinger KR, et al. STRIDES - STudying Risk to Improve DisparitiES in Cervical Cancer in Mississippi - Design and baseline results of a Statewide Cohort Study. Prev Med. 2021;153:106740. doi:10.1016/j.ypmed.2021.106740.
American Society for Colposcopy and Cervical Pathology. https://www.asccp.org/enduring-guidelines-process. Last accessed March 8, 2024.