Dear Editor,

Cervical cancer is the fourth most common malignancy in women worldwide and also the fourth leading cause of cancer mortality.[1] Majority of the cervical cancer cases are attributed to untreated human papillomavirus (HPV) infection. There are over 200 genotypes of HPV, but only a few of them are oncogenic. Although most HPV infections will resolve spontaneously within 12–24 months, persistent infections may lead to precancerous lesions. When these dysplastic lesions are left without surveillance or treatment for a long time, 10%–20% of them will result in invasive cervical cancer.[2] Cervical cytology has been a major breakthrough in the identification of cervical (pre) cancerous lesions and has led to a decrease in rates of cervical cancer worldwide; however, due to its suboptimal sensitivity, it is being increasingly replaced by HPV DNA testing as a primary modality for cervical cancer screening.[3] On the other hand, the latter presents decreased specificity, and it requires further triage, which currently is, in most cases, cytology. There are, though, novel triage tools, which could better facilitate risk stratification and colposcopy referral, to avoid overtreatment.

Recently, p16/Ki67 dual stain (DS) cytology has been proposed as a new triage tool for HPV infections instead of classic cervical cytology for HPV-positive women.[4-6] p16 and Ki67 proteins are related to cell-cycle regulation and cellular proliferation. Detection of both of them in the same cell indicates that the oncogenic transformation process has started and that cervical intraepithelial neoplasia (CIN) could be diagnosed in cervical biopsies.[7] In 2020, p16/Ki67 DS was approved by the Food and Drug Administration for the triage of HPV-positive women to colposcopy.

Many studies have investigated and validated DS high detection rate of CIN compared to cytology or co-testing. It all started in 2013, with a prospective multicenter European trial Primary ASC-US and LSIL Marker Study (PALMS study) with 27,349 women.[6] All patients underwent cervical cytology, HPV testing, and DS, and CIN2+ was set as the disease detection cutoff. The analysis showed that DS had superior sensitivity and comparable specificity to cervical cytology for detecting CIN2+, especially for women under 30 years old. Furthermore, an exploratory subset data analysis (with DS testing) of the prospective Addressing the Need for Advanced HPV Diagnostics (ATHENA) trial[5] with 7,727 patients showed similar results. DS has a higher detection rate of CIN2+ and CIN3+ cases compared to cytology, with fewer colposcopy referrals, even after HPV genotyping. These results reinforced DS as a superior triage tool for HPV-positive women. However, the cornerstone for the establishment of DS was a multicenter prospective IMproved Primary screening And Colposcopy Triage (IMPACT) trial from the USA, which included 35,263 women.[4] The results of this study showed that DS is associated with better risk stratification than cervical cytology and provided high reassurance for the occurrence of precancerous lesions both at baseline and at 1-year follow-up, irrespective of the HPV genotype.

As the performance of p16/Ki67 DS for the detection of oncogenic transformation in HPV-positive women is being recognized worldwide, it is gradually being used in daily clinical practices in many countries. Recently, it has been incorporated in the American Society for Colposcopy and Cervical Pathology (ASCCP) guidelines by the Enduring Consensus Cervical Cancer Screening and Management Guidelines Committee.[8] For high-risk HPV-positive women with no genotyping, DS is an acceptable option. In case of a positive result, colposcopy is recommended, while for a negative result, a 1-year follow-up is proposed. For HPV testing with partial HPV (16/18) genotyping, DS is also acceptable. Women with HPV 16/18 positivity should be referred for colposcopy irrespective of DS result. However, women testing positive for other high-risk HPVs (hrHPVs) should undergo colposcopy, in case of a positive DS; those with negative DS should repeat testing after 1-year. Moreover, in the co-testing setting, DS is acceptable for HPV-positive women with cytology results negative for intraepithelial lesion malignancy, atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesion. When genotyping is not available, a positive DS justifies colposcopy referral, and a negative one, 1-year follow-up. In case of atypical squamous cells – cannot rule out high-grade, atypical glandular cells – cannot rule out high-grade or high-grade squamous intraepithelial lesion, DS is not recommended and should not guide management.

Even though p16/Ki67 DS has been established as a new triage tool for the HPV precancerous cervical lesions. The majority of CIN1+ lesions will spontaneously regress without any further treatment. In case of CIN3+, the risk of cervical carcinogenesis is high and demands surgical treatment. CIN2+ is an ambiguous diagnosis that, although predisposes for the progression of cervical carcinogenesis, it is associated with a high-regression rate. CIN2+ and CIN3+ are considered high-grade lesions. Hence, in case of the management of CIN2+ other factors should be considered, such as age or future pregnancy plans. Currently, excisional treatment tends to be avoided in cases of young women at childbearing age, with CIN2+, because it has been linked with an increased risk of preterm birth in a subsequent pregnancy.[9] Therefore, there is a subset of women with CIN2+ lesions who wish to avoid excisional treatment because their concern for cervical cancer risk is less important than their concern for future adverse pregnancy outcomes.

Those women to date are closely monitored with cytology and colposcopy. There is an ongoing prospective trial.[10] which investigates the use of p16/Ki67 DS for the monitoring of these women to better detect possible lesion progression, minimizing unnecessary colposcopy referrals. The hypothesis of the study is that p16/Ki-67 DS can accurately detect progression to CIN3+ in women with CIN2+ who have not been subjected to excisional treatment.

In conclusion, the use of p16/Ki67 DS as a triage tool can result in earlier detection of precancerous cervical lesions with fewer colposcopy referrals. This relatively novel modality may also have other uses in the cervical cancer screening and management pathway.

Not applicable.

AGC: Atypical glandular cells – cannot rule out high grade

ASC-H: Atypical squamous cells – cannot rule out high grade

ASCUS: Atypical squamous cells of undetermined significance

CIN: Cervical intraepithelial neoplasia

DS: p16/Ki67 dual stain

HPV: Human Papilloma Virus

HSIL: High-grade squamous intraepithelial lesion

LSIL: Low-grade squamous intraepithelial lesion

NILM: Negative for intraepithelial lesion malignancy

DZ and DT: Designed the study; all authors conducted the study; DZ, KC, and DT: Participated in drafting the manuscript, and all authors contributed to critical revision of the manuscript for important intellectual content; DT: Supervised the study. All authors participated fully in the work, took public responsibility for appropriate portions of the content, and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or completeness of any part of the work were appropriately investigated and resolved. All authors gave final approval of the version to be published. All authors meet ICMJE authorship requirements.