| Background:With the popularization of cervical cancer screening, the number of high-grade cervical intraepithelial neoplasia (CIN) is increasing gradually. Cervical conization can be used for high-grade CIN and effectively reduce the occurrence of cervical cancer. But some patients may have residual or recurrent lesions after cervical conization and the risk of developing into cervical cancer is higher than that of ordinary people. The follow-up and managements after conization are key problems to prevent and cure cervical cancer after preliminary screening. The persistent infection of high-risk HPV type after conization is a high-risk factor for the recurrence. However, there are no effective therapeutic approaches on HPV affection recently. According to 2012 ASCCP guidelines, cervical conization or hysterectomy can be performed for the treatment of persistent or recurrent high-grade CIN. But it’s not suitable to operate hysterectomy for those who have fertility requirements. And repeat cervical conization may result in cervical incompetence, abortion, premature delivery or other complications. Therefore, we need a method to reserve the cervix as well as cure cervical lesions. Photodynamic Therapy (PDT) is a kind of new minimally invasive therapy, and application of PDT in cervical lesions is still at the stage of exploration.The First Part:Clinical Diagnosis and Treatment of Residual or RecurrentLesions After Cervical Conization for Cervical Intraepithelial NeoplasiaObjectivesTo analyze the clinical value of cervical cytology TCT, HPV test, colposcopy and cervical biopsy in residual or recurrent lesions after cervical conization for CIN.MethodsThe patients who received colposcopy and conization of the cervix from January 2012 to July 2014 in Qilu Hospital, Shandong University were chosen, then their clinicpathological data were analyzed, and the diagnosis "gold standard" was based on histopathological results, and the final diagnosis was the worst diagnosis of cervical biopsy or ECC or surgical tissue. The sensitivity and specificity of cytology, HPV test and colposcopy were calculated, and the diagnosis of colposcopy and clinicpathological results were compared. Also, colposcopy-directed biopsy results were compared with the pathological results of second surgery.Results1.317 patients who received cervical conization were recruited in this study, and 355 colposcopy-directed biopsies were performed.The mean age of patients was (41.1±7.8) years old. Based on the worst pathological results of the biopsy or the second surgery,145 cases were normal or cervicitis, and 90 cases were LSIL, and 118 cases were HSIL, and 2 cases were microinvasive carcinoma.2. The sensitivity of TCT, HPV test and colposcopy in diagnosing HSIL+(HSIL and invasive carcinoma) were respectively 69.1%(58.4%-79.8%),96.7%(92.5%-100.0%),61.7%(55.8%-67.5%); the specificity is 65.6%(59.9%-71.3%), 13.9%(9.8%-18.0%),95.7%(93.3%-98.2%). The sensitivity of TCT, HPV test and colposcopy in diagnosing SIL+(LSIL and more severe lesion) is 57.6% (50.7%-64.5%),93.8%(90.5%-97.2%),76.7%(71.6%-81.7%); the specificity is respectively 70.7%(65,3%-76.1%),16.4%(12.0%-20.8%),63.4%(57.7%-69.2%).3. The complete agreement of colposcopic diagnosis and the final pathological results was 59.7%(212/355), and the strength of areement value Kappa is 0.40 (P <0.05); the complete agreement between the different cervical transformation zone was respectively, type Ⅰ with 57.6%(76/132), type Ⅱ with 61.9%(60/97), type Ⅲ with 60.3%(76/126), and the difference was not significant. The agreement within one grade (the same diagnosis or within one grade) is 95.2% (338/355). The colposcopc diagnosis were 16.1%(57/355) overestimated, and 24.2%(86/355) were underdiagnosed. When the colposcopic diagnosis were normal or cevisitis, the complete agreement was 63.2%(84/133), the underdiagnosis rate was 36.8%(49/133); when the colposcopic diagnosis were LSIL, the complete agreement is 36.2%(47/130), the underdiagnosis rate was 27.7%(36/130); the differences were significant. Therefore, the complete agreement rate when the colposcopy diagnosis was normal or LSIL was lower than that when the colposcopy diagnosis was HSIL.4. ECC:There were total 156 ECC tests in 355 colpscopic examinations, of which 10.3%(16/156) of cervical canal tissue could not be got,8.3%(13/156) failed due to cervical canal stenosis.129 cases of cervix underwent pathological examination, and 85.8%(109/129) were NILM,1.6%(2/129) were LSIL and 12.6%(16/129) were HSIL. And 2.5%(3/120) HSIL cases were diagnosed based on ECC while cervical biopsies were negative.5.123 patients got the second surgery after conization of the cervix. Comparing the pathological results between colposcopy-directed biopsy of cercix (including ECC) and surgical tissue, rate of agreement was 55.3%(68/123),42.3%(52/123) of pathological results degraded after operation, and 2.4%(3/123) became more serious after operation. Two cases of microinvasive carcinoma were diagnosed after surgery and preoperative biopsy was LSIL and HSIL, respectively.6. Treatment:112 patients whose biopsy results were HSIL got repeat cervical conization or hysterectomy,4 patients whose biopsy results were HSIL took part in the clinical trial and used ALA-PDT to treat the lesions.11 patients whose biopsy results were LSIL got repeat cervical conization. And 2 patients who infected high risk HPV for more than one year took part in theALA-PDT clinical trial.Conclusions1. In diagnosing residual or resurrent lesions after cervical conization, the sensitivity of HPV test was relatively high, and the specificity of TCT is relatively high. Some recurrent high-grade CIN may miss diagnosis if referral to colposcopy only depended on TCT results.2. When colposcopic diagnosis was normal or LSIL in patients who had cervical conization, there was the risk of underdiagonis. Some persistant or recurrent HISL may miss diagnosis if cervical biopsy were only depended on positive colposcopic results.3. ECC may help to improve the detection rate of residual or recurrent lesions after conization of the cervix.4. The application of PDT in treating the persistant or resurrent lesions after cervical conization needs more research.The Second Part:The Preliminary Exploration of Biological Effects and Mechanism of PDT on Human Immortal Cervical Cell Line H8ObjectivesTo observe the biological effects of PDT based on new water-soluble photosensitizer TMPyP on H8 cell line (HPV16 E6/E7 gene immortalized human cervical epithelium cell), and to explore their possible mechanisms.MethodsThe morphology of H8 cell after TMPyP-PDT was observed under the inverted microscope; the localization of photosensitizer in the cell was observe by the fluorescence microscope. The cellular proliferative activity were detected using the CCK-8 assay kit. The cell apoptosis were analysed using AnnexinV-FITC/PI flow cytometry kit. The change of p16INK4a/Ki67 caused vy TMPyP-PDT on H8 was detected by the immunochemistry SABC method. The expression of ERK、JNK、 hTERT、p21 and p16INK4a were analysed by Western Blotting.ResultsThe photosensitizer TMPyP was mainly distributed in the nucleus and TMPyP-PDT changed the morphology of H8 cell, suppressed the cell proliferation and reduce the cell survival rate, and its effects were related to the concentration of TMPyP. TMPyP-PDT induced the apoptosis of H8 cell. Immunocytochemistry analysis showed that TMPyP-PDT reduced the expression of p16INK4a in H8 cell; Western Blotting showed that TMPyP-PDT reduced the phosphorylation level of ERK and JNK, and decreased the expression of hTERT and p16INK4a, but increased the expression of protein p21.ConclusionTMPyP-PDT could inhibit the proliferation of H8, and induced the apoptosis of H8, decreased the expression of protein p16INK4a/Ki67. The mechanism may be related to the downregulation of ERK and JNK phosphorylation, the decreased of hTERT and p16INK4a as well as upregulation of p21. |
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