Study Of Cervical Transformation Zone In The Diagnosis And Treatment Of Cervical Intraepithelial Neoplasia And Mechanism Of Cervical Canal Depth

BackgroundCervical intraepithelial neoplasia(CIN)is the precancerous lesion of cervical cancer classified into grade 1,2 and 3 according to the degree of abnormal cell proliferation and differentiation.In 2014,the 4th edition of the WHO classification of female genital tumors classified cervical precancerous lesions of squamous cell into two grades,low-grade squamous intraepithelial lesion(LSIL)which was CIN1 and high-grade squamous lesion(HSIL)which was CIN2 and CIN3.The classification in the 5th edition of WHO classification of female genital tumors in September 2020 had not changed,and was still LSIL and HSIL.However,for more precise management,it was recommended to distinguish CIN2 and CIN3 as much as possible when diagnosing HSIL.CIN reflects the continuous process in the occurrence and development of cervical cancer.Untreated HSIL had the 31%to 50%risk of progressing to cervical cancer within 30 years,while treated HSIL had the 0.7%risk.Therefore,screening and treatment of HSIL can reduce the incidence of cervical cancer.As the important means of cervical cancer screening,colposcopy plays a bridge role from the abnormal initial screening results to the diagnosis of cervical intraepithelial neoplasia and guiding treatment.Therefore,standardizing the colposcopy evaluation has always been the subject of concern and discussion.As the world’s authoritative organization in the field,the International Federation of Cervical Pathology and Colposcopy(IFCPC)and the American Society for Colposcopy and Cervical Pathology(ASCCP)regularly update the guidelines for colposcopy in order to improve the standardization of colposcopy and ensure that it plays its due value in the prevention of cervical cancer.The transformation zone(TZ)of the cervix has always been the important assessment of colposcopy,and most precancerous lesions of the cervix occur in the transformation zone.The 2011 IFCPC colposcopy terminology also recommend to perform type 1,2,and 3 excision to correspond to the 1,2,and 3 transformation zone.However,in 2017,ASCCP claimed that literature suggested the use of TZ type unrepeatable,especially for type 2 TZ,and there was no evidence showed TZ type could improve the prediction or management of cervical disease.Therefore,TZ types were not incorporated in the 2017 ASCCP terminology.We believe that the existence of TZ depends on whether it has a guiding role in the diagnosis and treatment of cervical intraepithelial neoplasia(CIN).If evidence-based research suggests that the TZ has clinical significance,further effort to reduce heterogeneity in the classification of TZ types between individual examiners is of importance,instead of denying it because of poor repeatability.To verify the TZ’s value of diagnosis and treatment,the study analyzed whether TZ had an impact on the accuracy of colposcopy diagnosis and whether there was a difference in the depth of cervical canal resection of loop electrosurgical excision procedure(LEEP).We also studied the diagnostic value of the 2011 IFCPC colposcopy terminology.LEEP is widely applied in the treatment of high grade squamous intraepithelial lesion(HSIL).However,there is no other objective indicator to guide the depth of cervical canal resection besides the type of TZ.When we analyzed clinical data,we found that even in cases of the same type TZ,the depths of the lesion involving the cervical canal were different.Therefore,we performed proteomic analysis on the depth of cervical squamous intraepithelial lesions.It is expected that these studies will provide clinical data with certain guiding significance for the diagnosis and treatment of cervical precancerous lesions.Part Ⅰ The Impact of Different Types of Cervical Transformation Zone in the Colposcopic Diagnosis of Cervical Intraepithelial Neoplasia andClinical Application of the 2011 IFCPC Colposcope TerminologyObjective:To explore the impact of different types of cervical transformation zone in the colposcopic diagnosis of cervical intraepithelial neoplasia.To evaluate colposcopic accuracy according to the 2011 International Federation of Cervical Pathology and Colposcopy(IFCPC)terminology.Methods:A retrospective study of 1,838 patients was carried out.All patients underwent colposcopy in Shangdong Provincal Qianfoshan Hospital from October 2013 to April 2018.Targeted biopsies were taken from the most severe abnormal areas.Patients received colposcopic diagnoses according to the 2011 IFCPC colposcopic terminology.Colposcopic diagnoses were classified as normal or benign,LSIL,HSIL and invasive carcinoma.According to the pathological diagnoses of cervical biopsy,Patients with indications for surgery underwent further cervical conization,or hysterectomy after conization,combined with cervical biopsy pathology and postoperative pathology,and the most severe pathological diagnosis was taken as the final diagnosis.Pathological diagnosis was divided into:normal or benign,LSIL,HSIL,and carcinoma.Explore the impact of different types of cervical transformation zone in the colposcopic diagnosis,analyze the value of the 2011 IFCPC terminology.Results:1.The accuracy of colposcopic diagnosis of type 1,2 and 3 transformation zones were 60.8%,56%and 66.1%,respectively.The difference was not statistically significant(P=0.134).There was no significant difference in the accuracy of colposcopic diagnosis in type 1,2 and 3 transformation zones in the normal or benign group,LSIL,HSIL and carcinoma group.2.Data from 1,838 patients were analyzed in this study.The colposcopic diagnosis and histological diagnosis were consistent in 1,196 cases(65.1%)with a weighted kappa=0.60(95%CI=0.57～0.63,P<0.001).3.The sensitivity,specificity,PPV,NPV and YI of thin acetowhite epithelium detecting LSIL were 87.03%,59.02%,53.15%,89.49%and 0.4605.The sensitivity,specificity,PPV,NPV and YI of dense acetowhite epithelium in the diagnosis of HSIL were 73.22%,87.87%,70.37%,89.29%,0.6109,respectively.4.Logistics regression analysis showed the odds ratio(OR)of mustard yellow for HSIL was 1.43(95%CI 1.06～1.94,P=0.019).Conclusions:1.Cervical transformation zone types may have no impact on the accuracy of the colposcopic diagnosis.2.The acetowhite epithelium still has important diagnostic value,however,the value of a few signs is needed to be discussed and new signs are expected to be discovered.3.Although the significance of Lugol’s staining was diminishing,mustard yellow might be a valuable indicator for the diagnosis of HSIL.Part Ⅱ The Impact of Different Types of Cervical Transformation Zone in the Length of LEEP Resection and the High-risk Factors Associated with Positive Endocervical MarginsObjective:To evaluate the impact of different types of cervical transformation zone in the length of LEEP resection.To analysis the high-risk factors associated with positive endocervical margins.Methods:516 patients underwent colposcopy and targeted biopsies with the pathological diagnosis of HSIL and HSIL not excluding stage IA1 cervical cancer in Shangdong Qianfoshan Hospital,from October 2013 to October 2020.All patients underwent LEEP and had the pathological diagnosis with the information on length,thickness,circumference and cone margin status of the cone specimen recorded.Combined with cervical biopsy pathology and conization pathology,the most severe pathological diagnosis was taken as the final diagnosis with the classification of benign,LSIL,HSIL and invasive carcinoma.Compare the length of LEEP resection in different cervical transformation zones.Logistic regression analysis was conducted to study the risk factors of positive endocervical margins of LEEP.Results:1.The data was comprised of 521 patients who underwent colposcopy and LEEP procedure because of CIN in Shangdong Qianfoshan Hospital.The final selected cases were 516.Pathologically diagnosed were 487 cases of HSIL and 29 cases of stage IA1 cervical cancer.There were 474 cases with negative endocervical margins of conization specimens and 42 cases with positive endocervical margins.2.In the negative endocervical margin group,the LEEP resection lengths of transformation zone type 1,2,and 3 were 1.66±0.72cm,1.47±0.6cm,1.71±0.67cm,respectively,and the difference was not statistically significant(P=0.053).As types 1 and 2 transformation zone are both "squamocolumnar junction completely visible," we further compared the LEEP resection lengths in type 1+2 transformation zone and type 3 transformation zone.The results showed that the resection length of the type 1+2 transformation zone was 1.57±0.67 cm,and the resection length of the type 3 transformation zone was 1.71±0.67 cm,the difference was statistically significant(P=0.039).3.In the negative endocervical margin group,451 cases were pathologically diagnosed as HSIL,and the length of LEEP resection was 1.66±0.67cm;23 cases were pathologically diagnosed as cervical cancer stage IA1,and the length of LEEP resection was 2.07±0.54 cm,the difference was statistically significant(P=0.001).4.We further performed the logistic regression analysis of age,resection length,type of transformation zone and lesion grade of the positive and negative endocervical margin groups.The results showed that the grade of the lesion was a risk factor for positive resection margins.The probability of positive resection margin of cervical cancer stage IAlwas 3.275 times than that of HSIL(OR value is 3.275,95%CI 1.254～8.558,P=0.015).Conclusions:1.Cervical transformation zone 1,2,3 types may have no impact on the LEEP resection length.If the type 1 and type 2 transformation zones are combined,the resection length of the type 1+2 transformation zone is less than that of the type 3 transformation zone.2.Compared with HSIL,LEEP for cervical cancer stage IA1 requires more cervical canal tissue to be removed.For highly suspicious invasive cervical cancer(such as cervical stage IA1)before surgery,LEEP requires more cervical canal tissue to be removed or cold knife conization could be recommended.3.A logistic regression analysis of age,resection length,type of transformation zone,and lesion grade(HSIL and cervical cancer stage IA1)indicates that the lesion grade(cervical cancer stage IA1)might be the risk factor for the positive endocervical margin of LEEP.Part Ⅲ Label-Free Quantitative Proteomic Analysis of the Cervical Canal Depth of Cervical Intraepithelial NeoplasiaObjective:To explore the proteins affecting the cervical canal depth of cervical intraepithelial neoplasia and guiding the length of LEEP resection.Methods:The specimens were formalin-fixed and paraffin-embedded cervical tissue provided by the Department of Pathology,Shangdong Provincal Qianfoshan Hospital.The control group(CK group)consisted of 3 cases of adenomyosis who underwent laparoscopic hysterectomy in the Department of Gynecology.The preoperative cervical screening of TCT and HPV were negative,and the postoperative cervical pathology was cervicitis.The experimental QQ group consisted of 3 HSIL cases with LEEP resection length of 0.8 cm;endocervical margins was negative which indicated the lesion in the cervical canal was superficial.The experimental SQ group consisted of 3 HSIL cases with LEEP resection length 2.57±0.40cm;endocervical margins was positive which indicated the lesion in the cervical canal was deep.Label-free quantitative proteomics technology was used for protein extraction and peptide digestion,LCMS/MS analysis,identification and quantitation of proteins,protein clustering analysis,subcellular localization analysis,Domain annotation,GO annotation,KEGG annotation,protein-protein interaction analysis.Enrichment analysis of GO annotation(or KEGG pathway,or Domain)annotation on the target proteins was applied based on the Fisher’ exact test.Nine cases in three groups were selected for parallel reaction monitoring(PRM)to verify the different proteins by proteomics.Fibronectin verified by PRM was immunohistochemically stained in the paraffin sections of previous cases to further analyze the clinical significance.Results:Using Fold Change(FC)up-regulation>2.0 times or down-regulation<0.5 times and P<0.05(T test or other)as the standard,there were 33 significant different proteins in SQ and QQ groups;83 in SQ and CK groups;81 in QQ and CK groups.Three candidate proteins of TNC,FN and CDH13 were verified by PRM to be consistent with proteomics.The positive expression rates of fibronectin immunohistochemically stained among CK-IHC group,QQIHC group and SQ-IHC group were 5%,30%and 65%,the difference was statistically significant(χ2=16.359,P=0.000).Conclusions:1.There are significant difference proteins in normal cervix group and superficial cervical canal lesion group,normal cervix group and deep cervical canal lesion group,superficial cervical canal lesion group and deep cervical canal lesion group.2.Fibronectin 1(FN1)and Tenascin C(TNC)associated with EMT may be proteins related to the cervical canal depth of HSIL.