The Significance Of P16/Ki67 Double Staining In The Prediction Of High-grade Cervical Lesions And Its Guiding Value In The Follow-up Of Low-grade Cervical Lesions

BackgroundCervical cancer is the second most dangerous cancer to women’s health worldwide after breast cancer,and the incidence of cervical cancer has been increasing in recent years.According to the statistical results released by the world health organization（WHO）,among the newly increased cervical cancer cases worldwide every year,China has reached 75,000 cases,ranking the second in the world in the incidence rate,and the incidence population shows an obvious trend of youth^[1].Although in recent years,cervical cancer incidence and mortality have been significantly reduced due to the popularization of cervical precancerous lesions screening and cervical cancer vaccine,there are still phenomena of over-diagnosis and under-diagnosis in clinical practice.Currently,the three-step method,namely pap smear cytology-colposcopy-cervical histopathology,is used in China,and its limitations have been exposed after years of application.Pap smear cytology test not only requires professional pathologists to observe the diagnosis under the microscope,if the infection and other factors caused by atypical cell morphology,there is likely to be a certain false negative and false positive rate,the sensitivity is low.Colposcopy and cervical histopathology are invasive tests that are not suitable for screening all patients.A large number of epidemiological and biological data have proved that persistent high-risk human papillomavirus（HR-HPV）infection can be a key pathogenic factor for cervical cancer and precancerous lesions,and is also an important factor for cervical cancer.HPV detection is also one of the main screening methods for cervical cancer and precancerous lesions recognized at home and abroad.In the prevention and control guidelines of cervical cancer issued by WHO in 2013,HPV detection is also explicitly recommended as the primary screening method for cervical cancer screening.However,its specificity is poor,and it cannot recognize whether it is transient infection or transformed infection,resulting in excessive colposcopy referrals.Therefore,it is still necessary to find a simple and accurate technique to predict high-grade cervical squamous intraepithelial lesions.At present,SIL is divided into two levels:low-grade squamous intraepithelial lesion（LSIL）and high-grade squamous intraepithelial lesion（HSIL）.Because of their different ending and outcomes,the principles of treatment for them are also different.LSIL is generally considered to be the histopathological representation of carcinogenic or non-carcinogenic human papillomavirus（HPV）infection,which requires clinical observation through follow-up.HSIL is a precancerous lesion that requires surgical treatment^[2].HSIL detection is critical in cervical cancer prevention.Now,in clinical follow-up of LSIL patients,the above indexes cannot predict the prognosis and outcome of the disease well,and there are few reports on the detection indexes of the prognosis and outcome.Therefore,it is of great significance to search for appropriate markers that can predict the progression and outcome of LSIL lesions to prevent the occurrence and development of cervical cancer.For a long time,cervical intraepithelial neoplasia（CIN）has been the main diagnostic name of SIL at home and abroad,including three grades,namely CIN I,CIN II and CIN III.In the fourth edition of classification of female genital tumors published by WHO in 2014,CINII grade lesions were not included in HSIL.In essence,CINII grade lesions were the mixed structure between CINI and CINIII in biology.If all CINII grade lesions are classified into HSIL,it may cause over treatment.At home and abroad,P16 is used to identify cervical specimens with CINII grade lesions which are difficult to diagnose.If P16 is positive,it can be diagnosed as HSIL;if P16 is negative,it can be diagnosed as LSIL.It can improve the accuracy and objectivity of pathological diagnosis,improve the accuracy of CIN classification,and reduce the over treatment or delayed treatment due to subjective judgment^[3-4].P16 is the first tumor suppressor gene,and it is also an objective index to distinguish LSIL and HSIL.It has been reported that P16 is also an important observation index to predict the recovery of SIL.It has been confirmed that HPV infection is the main cause of cervical precancerous lesions and cervical cancer.The overexpression of P16 protein shows that HPV infection leads to host cell mutation,suggesting that P16 may be an important molecular biological marker for the initiation of cervical carcinogenesis^[5-6].Ki67 is one of the most reliable indicators of cell proliferation activity.Although it is not involved in cervical carcinogenesis,it has good predictive significance for disease classification and evaluation.The higher CIN level,the more active cell proliferation and the stronger Ki67 expression.Under normal physiological conditions,P16 and Ki67 should be mutually exclusive.However,if P16 and Ki67 are over expressed in the same cell,it indicates that the cell cycle regulation is abnormal,which is of great significance for the detection of high-grade squamous intraepithelial lesions and cervical cancer.The positive rate of P16/Ki67 double staining increased with the aggravation of cervical disease.At the same time,P16/Ki67 has a certain clinical value in predicting the outcome of LSIL,to a certain extent,it can avoid the occurrence of over treatment in clinical work^[7].Objective:To study the significance of immunocytochemical P16/Ki67 double staining in the prediction of high-grade cervical squamous intraepithelial lesions and its guiding value in the follow-up of low-grade cervical squamous intraepithelial lesions.Methods:We collected patients who went to the Changning District Maternal and Child Health Care Hospital of Shanghai from December 2016 to November 2017 and voluntarily screened for cervical cancer.315 female patients with atypical squamous cells（ASCUS）and above diagnosed by liquid based cytology（LCT）were selected and high risk human papillomavirus（HR-HPV）were tested at the same time.Among them,11 cases were removed by unable to P16/Ki67 double staining because of insufficient cell quantity and unsatisfactory cytology.The final effective number of specimens was 304,all of them were detected by P16/Ki67 double staining,and colposcopy biopsy was taken as the gold standard.In addition,20 cases of liquid-based cytology were selected as negative for intraepithelial lesion or malignancy（NILM）for detection of P16/Ki67 double staining.90 patients with LSIL confirmed by colposcopy were followed up for 2 years.During the follow-up,high-risk HPV and TCT were detected and followed up once every half a year for 2years.Cervical biopsy and/or curettage under colposcope were performed when follow up to the 24th month.During the follow-up,if the biopsy pathology indicates HSIL or cervical cancer,the follow-up shall be terminated.HSIL were operated in time,and cervical cancer was hospitalized for further treatment.To analyze the value of P16/Ki67 double staining in the follow-up of low-grade cervical squamous intraepithelial lesions.Results:（1）The P16/Ki67 double staining results of 20 cases of no intraepithelial lesion cells or malignant cells（NILM）specimens were all negative.（2）The sensitivity,specificity,positive predictive value and negative predictive value of HSIL+（equivalent to CINII+）in ASCUS were 66.67%,95.92%,50.00%,97.92%,respectively.（3）The sensitivity,specificity,positive predictive value and negative predictive value of HSIL+（equivalent to CINII+）in LSIL cases were 91.89%,95.16%,91.89%and 95.16%respectively.（4）The sensitivity of HSIL+（equivalent to CINII+）was 93.48%,positive predictive value was 93.48%in HSIL（5）The sensitivity,specificity,positive predictive value and negative predictive value of HSIL+（equivalent to CINII+）in ASCUS and above were 90.22%,94.34%,87.37%and 95.69%,respectively.（6）A 2-year follow-up of 90 LSIL patients showed that the outcome of P16/Ki67double negative staining group was significantly better than that of the double positive staining group（P<0.05）.Conclusion:（1）Immunocytochemical P16/Ki67 double staining can significantly improve the predictive value of HSIL+detection in HR-HPV positive and LCT≥ASCUS patients,which is expected to be an effective auxiliary method for HPV screening.（2）It is of certain significance to predict the outcome of LSIL and can be used in follow-up shunt management of LSIL,which is worthy of clinical promotion and application.