Recently, the 20th Academic Meeting of the Pathology Branch of the Chinese Medical Association and the 4th Annual China Pathology Conference were held in Chongqing. During the meeting, Prof. Zhao Yun from Peking University People’s Hospital, Prof. Guo Huiqin from Cancer Hospital of Chinese Academy of Medical Sciences and Prof. Mei Ping from Guangdong Provincial People’s Hospital discussed the latest CINtec PLUS (p16/Ki-67 cytology double-staining test) and the application of p16 histology test in cervical cancer screening to effectively guide clinical practice. Concurrent p16/Ki-67 expression suggests dysregulation of cell cycle regulation Persistent human papillomavirus (HPV) infection is a major cause of cervical cancer. Usually, if HPV infection is not cleared, it becomes permanent and will produce viral oncogenic proteins E6 and E7, which can bind to the tumor suppressor protein p53 and the retinoblastoma protein pRb, respectively, inactivating both oncogenes. The HPV E6 protein has a high affinity for wild-type p53, and their binding can cause rapid degradation of p53, which can impede the cellular response to DNA damage and lead to the accumulation of genetic trait changes and consequently genotypic malignancies. HPV E7 protein interferes with the cell cycle, causing continued cell differentiation and precancerous lesions, leading to uncontrolled cell regulation, immortalization, and progression to cancer if left untreated. pRb, to which E7 protein binds preferentially, weakens pRb function, disrupts the ability of transcription factor E2F to bind, and starts the oncogenic transformation process, leading to deregulated epithelial cell growth, causing Ki-67, p16 protein The p16 gene is a cell cycle hormone-dependent gene. The p16 gene is a typical representative of cell cyclin-dependent kinase (CDK) inhibitors, which are associated with a variety of tumors and are called multiple tumor suppressor genes. p16 overexpression indicates that normal cells are in cell cycle arrest, and short duration of HPV infection does not affect the cell cycle regulation, while persistent The abnormal proliferation of HPV-infected cells leads to p16 overexpression, and therefore p16 overexpression can be used as a marker of cervical lesions. Ki-67 is a nuclear antigen gene that marks cell cycle progression and proliferative phase of cells, and its expression is restricted to G1, S, G2 and M phases of the cell proliferation cycle, and is absent in G0 phase. Ki-67 overexpression indicates that cells are in the proliferative phase of the cell cycle. Usually in physiologically normal cells, p16 and Ki-67 expression are antagonistic to each other and do not occur simultaneously. If p16 and Ki-67 are overexpressed at the same time, it indicates inactivation of pRb protein and dysregulation of cell cycle regulation. Therefore, simultaneous detection of p16 and Ki-67 in the same cell can be used as a marker of cell cycle dysregulation, which is associated with oncogenic transformation induced by high-risk HPV viruses and can help to detect truly diseased cells and is not dependent on morphological findings. When tested positive for p16/Ki-67, it strongly suggests high-grade lesions and provides an objective test to distinguish women with potentially high-grade lesions. In July 2012, the College of American Pathologists (CAP) and the American Society for Colposcopy and Cervical Pathology (ASCCP) guidelines stated that p16 can be used as a marker reflecting HPV E6/E7 affecting cell proliferation and that there is sufficient evidence for a recommendation related to low-grade anal-genital tract squamous epithelial lesions, recommending the use of p16INK4a antibody with a specific clone number (E6H4) as a biomarker to detect whether HPV infection affects cell cycle regulation. This clone number is the only IVD-approved p16INK4a antibody in the world. CINtec PLUS (p16/Ki-67 cytology double-staining test) improves cervical cancer screening triage management Currently, cervical cancer screening in China is facing problems such as low sensitivity of cytology testing (50-70%), poor inter-observer reproducibility, insufficient quality control and lack of technicians. The application of biomarker p16/Ki-67 can effectively improve cytology triage testing. The highly sensitive and specific CINtec PLUS test can be applied to: triage of atypical squamous epithelial cells without clear significance (ASC-US) (especially for young women); triage of low-grade squamous intraepithelial lesions (LSIL); triage of cytology-negative but high-risk HPV-positive individuals when combined cytology and HPV screening; and triage of high-risk HPV-positive individuals when HPV is used as primary screening CINtec PLUS technology can effectively reduce the underdiagnosis rate of high-grade cervical lesions, provide sufficient time and basis for clinical and patient care, and improve the detection and early intervention of pre-existing cervical disease. It can also reduce and avoid unnecessary colposcopic examinations. Professor Mei Ping from Guangdong Provincial People’s Hospital shared data from the CINtec PLUS clinical study at the meeting. A multi-country, multi-center prospective study PALMS (Primary ASCUS LSIL Marker Study) screened over 27,000 women with an average age of 39.9 years. All women were tested for traditional cytology (Pap smear), p16/Ki-67 double stain (CINtec PLUS) and HC2 HPV during the trial, with biopsy confirmation of CIN2+ as the endpoint of testing. The study confirmed that the CINtec PLUS test was 18% more sensitive for CIN2+ than conventional cytology without compromising specificity (86.7% vs 68.5%). For the triage of HPV-positive women, the CINtec PLUS double-stained test was 17% more sensitive for CIN3 compared to conventional cytology (88% vs 71%). Of the 575 patients with ASC-US on cytology, 18 patients had biopsy-confirmed CIN2 or higher, and CINtec PLUS double-staining had 94% sensitivity and 78% specificity, and was higher than HC2 HPV testing. Meanwhile, 63 of the 526 patients with LSIL were biopsy confirmed to have CIN2 or higher, and the specificity of the CINtec PLUS test was 54%, which was much higher than that of the HC2 HPV test. In addition, the EEMAPS (European Equivocal or Mildly Abnormal Papanicolaou Cytology) retrospective study performed biopsies, p16/Ki-67 double staining (CINtec Plus) and HC2 HPV testing on a total of 776 ASC-US/LSIL liquid-based cytology (ThinPrep) residual specimens from 5 European cytology laboratories within 6 months of cytology sampling. Among them, 77 of 361 ASC-US patients were confirmed to have CIN2 or more, and 72 of 425 LSIL patients were confirmed to have CIN3 or more. The sensitivity of CINtec PLUS test for CIN3+ was 96%, while the specificity was 68%, which was 49% higher than that of HC2 HPV test, indicating that the double-staining test can be used as a more reasonable means of LSIL triage. When combined cytology and HPV screening is used, the CINtec PLUS dual-stain test can be applied to triage those who are cytology-negative but high-risk HPV-positive. Results of a trial enrolling 425 women over 30 years of age who were cytology negative and tested positive for high-risk HPV showed that the CINtec PLUS dual-stain test had a sensitivity of 91.9% and specificity of 82.1% for CIN2+ and 96.4% and specificity of 76.9% for CIN3+. Professor Guo Huaiqin from the Cancer Hospital of the Academy of Medical Sciences also shared the experience of reading CINtec PLUS, which can effectively reduce the burden of the reading physicians by double-staining the films. In addition, for cases where morphological diagnosis is difficult, such as whether some crowded cell clusters are HSIL or reactive changes, the double-staining test can play an auxiliary role in diagnosis. The Roche Diagnostics CINtec PLUS Cytology test enables the combined detection of p16 and Ki-67 with excellent specificity and sensitivity. p16 signal (brown) and Ki-67 signal (red) in the same cell can be judged as a positive result by the simultaneous presence of brown staining of the cytoplasm and red staining of the nucleus. The test is expected to be available in China by the end of 2015. p16 histological test: improving the accuracy of pathological diagnosis and guiding clinical diagnosis of cervical cancer Professor Zhao Yun from Peking University People’s Hospital discussed the significance of the use of p16 in clinical practice from a clinician’s perspective. In particular, Prof. Zhao emphasized the damage caused to patients by over-treatment of cervical lesions, and therefore the clinical need for accurate pathological diagnostic results to guide patients’ treatment. Usually, cervical squamous epithelial precancerous lesions, i.e. cervical intraepithelial neoplasia (CIN), are classified into CIN1, CIN2 and CIN3 according to the degree of lesions, and clinical treatment strategies vary according to the different degrees of lesions. A more accurate grading of CIN is essential for clinical treatment decisions. Although the interpretation of H&E (Hematoxylin-eosin staining) staining histomorphology is the current standard for grading CIN, it is susceptible to the subjective judgment of pathologists, and is particularly poorly reproducible for CIN2. As basic and clinical research continues, the relationship between HPV and cervical cancer is becoming clearer, and it is gradually recognized that HPV exists in the free state in cervical cells, usually causing low-grade cervical lesions (LSIL), which have a very low risk of further progression to cervical cancer and are not true precancerous lesions; when it exists in the integrated form in host cells, it causes high-grade cervical lesions (HSIL), which are true precancerous lesions. This is a true precancerous lesion with a high risk of progression to cervical cancer. Based on this, the American Academy of Pathology (CAP) and the American Society for Colposcopy and Pathology (ASCCP) jointly published a consensus (LAST Project) recommending a two-tier nomenclature for cervical squamous intraepithelial lesions, divided into low-grade and high-grade lesions. The former includes CIN1 in the three-level nomenclature, and the latter includes CIN2 and CIN3. The fourth edition of the WHO classification of female genital tumors published in 2014 also recommends this nomenclature. However, in practice, the consistency of H&E diagnosis of CIN2 is low, and even in the United States, where pathologists are strictly trained, there is a large disagreement among physicians on the diagnosis of the same case. In fact, some of CIN2 is a true high-grade lesion, and a significant proportion is a low-grade lesion. When morphologic identification of high-grade or low-grade lesions is controversial, biomarkers are recommended to aid in the differential diagnosis. The LAST program has confirmed that the use of p16INK4a provides high-quality evidence for improved diagnostic consistency, and recommends the use of p16INK4a staining in four situations: when the differential diagnosis of HSIL and similar non-neoplastic lesions is required, such as immature squamous, atrophic, reparative epithelial hyperplasia and artifacts due to manual manipulation; questionable CIN2; differing diagnostic opinions between reviewers; HPV testing, cytology, and colposcopy suggesting the possibility of high-grade lesions, but a negative histologic diagnosis. Based on data from the LAST project, in 2014 WHO also recommended that for questionable diagnoses, p16INK4a immunohistochemical staining can be used to improve the accuracy of histological diagnosis of cervical lesions and the consistency of diagnosis between pathologists. The CAP and ASCCP recommend the same management of LSIL as CIN1 and HSIL, for most women, as CIN2/3. CIN2/p16INK4a-negative cases are managed as low-grade lesions (LSIL) with follow-up; CIN2/p16INK4a-positive cases are managed as low-grade lesions with follow-up. p16INK4a-positive cases were managed as high-grade lesions (HSIL) and treatment was recommended. According to the SEER study, the incidence of cervical cancer in women under 25 years of age is only 1.5/100,000. therefore, in young women with HSIL (CIN2) or HSIL without definite CIN2 or 3, cytology and colposcopy can be performed every 6 months and treatment is given if the lesion persists for 24 months, if colposcopy is unsatisfactory, if the lesion size increases or if colposcopy is worse than previous aggravation, then treatment is given. The biomarker p16INK4a enables a more microscopic understanding of the pathological mechanisms of cervical precancerous lesions, improves the accuracy of LSIL/HSIL interpretation, and ensures more rational clinical management of patients in triage. The Roche Diagnostics CINtec histology p16 test (containing anti-p16INK4a antibody (E6H4)) was launched in China in May 2014.