p16INK4A flow cytometry of exfoliated cervical cells: Its role in quantitative pathology and clinical diagnosis of squamous intraepithelial lesions

Author:

He Yifeng123,Shi Jun1,Zhao Hui4,Wang Yuefei56,Zhang Chi4,Han Sai7,He Qizhi8,Li Xiaolan9,Li Shangji1,Wang Wenjing2,Yi Muhua10,Hu Xiaoling11,Xing Zhihua12,Han Hao12,Gao Yinshuang12,Zhou Qing13,Lu Linlin14,Guo Jianfen15,Cao Hui16,Lu Caiping16,Hou Yanqiang16,Chen Dan17,Yang Fengyun18,Lei Ping19,Di Wen1220,Qian Ji21,Xia Yi16,Zhang Youzhong7,Deng Yang4,Zhu Jianlong3,Xu Congjian56

Affiliation:

1. Department of Obstetrics and Gynecology Ren Ji Hospital School of Medicine Shanghai Jiao Tong University Shanghai China

2. Shanghai Key Laboratory of Gynecologic Oncology Ren Ji Hospital School of Medicine Shanghai Jiao Tong University Shanghai China

3. Department of Obstetrics and Gynecology Pudong Hospital Fudan University Shanghai China

4. Department of Gynecology Taiyuan Maternal and Child Health Hospital Taiyuan Shanxi China

5. Department of Gynecology Obstetrics and Gynecology Hospital Fudan University Shanghai China

6. Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases Shanghai China

7. Department of Obstetrics and Gynecology Qilu Hospital Shandong University Jinan Shandong China

8. Department of Pathology First Maternity and Infant Health Hospital Tongji University Shanghai China

9. Department of Obstetrics and Gynecology The Second People's Hospital, Three Gorges University Yichang Hubei China

10. Department of Pathology Dongguan Hospital Southern Medical University Dongguan Guangdong China

11. Department of Obstetrics and Gynecology Yongcheng People's Hospital Yongcheng Henan China

12. Department of Obstetrics and Gynecology Zouping People's Hospital Zouping Shandong China

13. Department of Pathology The Central Hospital of Zibo Mining Group Co. Ltd. Zibo Shandong China

14. Department of Obstetrics and Gynecology The Central Hospital of Zibo Mining Group Co. Ltd. Zibo Shandong China

15. Department of Obstetrics and Gynecology Chifeng College Affiliated Hospital Chifeng Inner Mongolia China

16. Department of Clinical Laboratory Songjiang Hospital, School of Medicine, Shanghai Jiao Tong University Shanghai China

17. Fosun Diagnostic Technology (Shanghai) Co., Ltd Shanghai China

18. Department of Cervical Diseases Jiading Maternal and Child Health Care Hospital Shanghai China

19. Department of Gynecology Zhuhai Center for Maternal and Child Health Care Zhuhai Guangdong China

20. State Key Laboratory of Oncogene and Related Genes Shanghai Cancer Institute Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University Shanghai China

21. State Key Laboratory of Genetic Engineering Institute of Genetics, School of Life Sciences, Fudan University Shanghai China

Abstract

AbstractBackgroundP16INK4A is a surrogate signature compensating for the specificity and/or sensitivity deficiencies of the human papillomavirus (HPV) DNA and Papanicolaou smear (Pap) co‐test for detecting high‐grade cervical squamous intraepithelial lesions or worse (HSIL+). However, traditional p16INK4A immunostaining is labour intensive and skill demanding, and subjective biases cannot be avoided. Herein, we created a high‐throughput, quantitative diagnostic device, p16INK4A flow cytometry (FCM) and assessed its performances in cervical cancer screening and prevention.MethodsP16INK4A FCM was built upon a novel antibody clone and a series of positive and negative (p16INK4A‐knockout) standards. Since 2018, 24 100‐women (HPV‐positive/‐negative, Pap‐normal/‐abnormal) have been enrolled nationwide for two‐tier validation work. In cross‐sectional studies, age‐ and viral genotype‐dependent expression of p16INK4A was investigated, and optimal diagnostic parameter cut‐offs (using colposcopy and biopsy as a gold standard) were obtained. In cohort studies, the 2‐year prognostic values of p16INK4A were investigated with other risk factors by multivariate regression analyses in three cervicopathological conditions: HPV‐positive Pap‐normal, Pap‐abnormal biopsy‐negative and biopsy‐confirmed LSIL.ResultsP16INK4A FCM detected a minimal ratio of 0.01% positive cells. The p16INK4A‐positive ratio was 13.9 ± 1.8% among HPV‐negative NILM women and peaked at the ages of 40–49 years; after HPV infection, the ratio increased to 15.1 ± 1.6%, varying with the carcinogenesis of the viral genotype. Further increments were found in women with neoplastic lesions (HPV‐negative: 17.7 ± 5.0–21.4 ± 7.2%; HPV‐positive: 18.0 ± 5.2–20.0 ± 9.9%). Extremely low expression of p16INK4A was observed in women with HSILs. As the HPV‐combined double‐cut‐off‐ratio criterion was adopted, a Youden's index of 0.78 was obtained, which was significantly higher than that (0.72) of the HPV and Pap co‐test. The p16INK4A‐abnormal situation was an independent HSIL+ risk factor for 2‐year outcomes in all three cervicopathological conditions investigated (hazard ratios: 4.3–7.2).ConclusionsFCM‐based p16INK4A quantification offers a better choice for conveniently and precisely monitoring the occurrence of HSIL+ and directing risk‐stratification‐based interventions.

Funder

Science and Technology Commission of Shanghai Municipality

National Natural Science Foundation of China

Publisher

Wiley

Subject

Molecular Medicine,Medicine (miscellaneous)

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