[The value of P16/ki67 double labeling, HPV E6/E7 mRNA detection and their combined application in cytological shunt diagnosis of low-grade squamous intraepithelial lesions].

Objective To explore the value of double labeling of P16/ki67, E6/E7 mRNA of human papillomavirus (HPV) and combined detection in shunt diagnosis of low-grade squamous intraepithelial lesions (LSIL) by thin-layer cervical cytology (TCT). Methods The study enrolled 239 patients who underwent colposcopy and biopsy within 4 weeks after primary TCT diagnosis. The remaining cytological samples were double-labeled with P16/ki67 immunocytochemical staining and the HPV E6/E7 mRNA was detected by Panther automatic HPV E6/E7 mRNA detection system. Using SPSS22.0 software, the positive rates of P16/ki67 double-labeling, HPV E6/E7 mRNA and combined detection were analyzed in different cervical lesions, and the positive rates in the same cervical lesions were compared horizontally to evaluate the efficiency of double labeling of P16/ki67, HPV E6/E7 and combined detection in the diagnosis of high-grade squamous intraepithelial lesions (HSIL) and above lesions. Results The diagnostic results of HE staining for the 239 cases of LSIL were 71 cases of chronic cervicitis (29.71%), 143 cases of LSIL (59.83%), 22 cases of HSIL (9.20%) and 3 cases of cervical cancer (1.26%). There were 46 cases of P16+ki67+ lesions (19.25%), 41 cases of ki67+P16- lesions (17.15%), 33 cases of ki67-P16+ lesions (13.81%) and 119 cases of P16-ki67- lesions (49.79%). The positive rates of P16/ki67 double-labeling, HPV E6/E7 mRNA and combined detection increased with the severity of cervical lesions. The positive rate of combined detection was the highest in the HSIL lesions, which was higher than that of P16/ki67 double-labeling and HPV E6/E7 mRNA detection. The sensitivity of combined detection was higher than that of P16/ki67 double-labeling and HPV E6/E7 mRNA detection. The Youden index of joint detection was 0.7850. Conclusion The combined detection of P16/ki67 double labeling, HPV E6/E7 mRNA and HPV E6/E7 mRNA had a certain clinical value in the management of cell LSIL shunt diagnosis. The combined detection significantly improved the sensitivity and Youden index of HSIL and above lesions, while maintaining a high specificity and coincidence rate.

[Clinical Value of Peripheral Blood Circulating Tumor Cells Detection in Early Cervical Infiltrating Carcinoma Screening].

OBJECTIVE: To observe the difference of circulating tumor cells (CTC) in peripheral blood of patients with different degrees of cervical lesions, and to evaluate the effectiveness of CTC detection in screening early invasive cervical cancer. METHODS: From December 2015 to October 2017, 63 cases of cervicitis, low-grade and high-grade intraepithelial lesions (LSIL, HSIL) and early invasive cervical cancer were confirmed by histopathological and clinical stages in Zhongshan Boai Hospital and Zhongshan Hospital Affiliated to Zhongshan University. The immunomagnetic bead negative enrichment technique combined with immunofluorescence was used. In situ hybridization (imFISH) was used to detect CTC in peripheral blood of patients. The positive rate and quantity of CTC in four groups were analyzed. The diagnostic efficacy of CTC in early invasive cervical cancer was evaluated based on the results of histopathological diagnosis and clinical staging. RESULTS: ①The positive rates of CTC in cervicitis group, LSIL group, HSIL group and early invasive cervical cancer group were 0, 0, 19.05% and 84.13% respectively. The overall difference was statistically significant ( χ 2=504.00， P<0.05). The positive rate of CTC in early invasive cervical cancer group was higher than that in other groups ( P<0.008 3). The positive rates of CTC in HSIL group were significantly different from those in LSIL group and cervicitis group ( P<0.008 3). ②The average number (median) of CTC positive in cervicitis group, LSIL group, HSIL group and early invasive cervical cancer group was 0, 0, 1/4 mL, 3/4 mL, respectively. The average number of positive CTC in early invasive cervical cancer group was higher than that in other groups, and the difference was significant compared with other groups ( P<0.008 3). The average number of CTC positive in HSIL group was significantly different from that in LSIL and cervicitis group ( P<0.008 3). ③The sensitivity, specificity, coincidence rate, positive predictive value and negative predictive value of CTC positive results in the diagnosis of early invasive cervical cancer were 84.13%, 93.65%, 91.27%, 81.54% and 94.65%, respectively. CONCLUSION: CTC exists in patients with HSIL and early invasive cervical cancer. With the aggravation of cervical lesions, the positive rate and number of CTC test results increase. CTC detection in early invasive cervical cancer screening has a certain practical value in clinic.

[Paraffin section thickness in immunohistochemical detection of p16 expression in cervical tissue samples].

OBJECTIVE: To investigate paraffin section thickness in immunohistochemical detection of p16 expression in cervical tissue samples. METHODS: p16 immunohistochemical staining was performed in 150 cases of chronic cervicitis, 126 cases of low-grade squamous intraepithelial lesions(LSIL), 96 cases of high-grade squamous intraepithelial lesions (HSIL) and 78 cases of cervical cancer from January 2014 to March 2018 in Zhongshan Boai Hospital. The results of p16 protein expression in paraffin sections with thickness of 2, 3, 4, 5 and 6 µm were compared using Logistic regression analysis. RESULTS: With the increase of slice thickness, the staining of p16 protein in nucleus gradually increased. The thickness of cervical slices in chronic cervicitis and cervical cancer samples had no significant effect on the positive rate of p16 protein(χ2=7.817 and 1.332, both P>0.05), while the thickness of slices in LSIL and HSIL samples had significant effect on the positive rate of p16 protein (χ2=17.688 and 10.182, P<0.05 or P<0.01). The stable and reliable results were obtained when the slices were between 3 and 5 µm thick. CONCLUSIONS: Paraffin sections with thickness of 3.0-5.0 µm are recommended for immnohistochemical staining of p16 protein in cervical tissue samples.

[Volume of 4% neutral buffered formaldehyde affects the results of HER2 gene detected by fluorescence in situ hybridization in primary invasive breast cancer].

OBJECTIVE: To investigate the effects of the volume of 4% neutral phosphate buffered formalin fixative solution on the detection of human epidermal growth factor receptor 2 (HER2) gene by fluorescence in situ hybridization (FISH) in primary invasive breast cancer. METHODS: Tissue samples were collected from 109 patients with primary invasive breast cancer admitted in Zhongshan Boai Hospital from June 2014 to October 2016. The ratios of 4% phosphate buffered formalin fixative solution to sample volume samples were 3:1, 6:1, 9:1, 10:1, 15:1, 20:1 or 25:1 (groups A, B, C, D, E, F and G), respectively. Paraffin sections were made after 15 h of fixation. The amplification of HER2 gene was detected by FISH. The gene amplification results of HER2 were observed and compared in different groups. RESULTS: Fluorescence microscope showed that the tissue contour in groups A, B and C was vague, cell debris appeared, and the probe was positioned poorly; while the tissue contour was clear and complete in groups D, E, F and G and the probe was positioned accurately. The positive rate of HER2 was gradually increased from group A to D(χ2=8.601, P<0.01), and that remained stable at 24.77% in groups D to G. The positive rate of gene amplification in groups D, E, F and G was significantly higher than that in groups A, B and C (all P<0.05). CONCLUSIONS: When using FISH to detect HER2 gene in samples of primary breast invasive carcinoma, the volume of fixative solution should be at least 10 times of the sample volume to obtain accurate and stable results.

[Application of Van-Clear and xylene in the detection of cervical hTERC gene by fluorescence in situ hybridization].

OBJECTIVE: To observe the effect of Van-Clear on vamplification of human telomerase RNA component (hTERC) gene in cervical tissues by fluorescence in situ hybridization, and to determine the potential for Van-Clear to replace xylene.
 METHODS: A total of 278 specimens of cervix uteri were collected from inpatients of Department of Gynaecology in Boai Hospital of Zhongshan from January to February, 2015, with 81 cases of normal specimens, 68 cases of cervical intraepithelial neoplasia (CIN) I, 57cases of CIN2, 42 cases of CIN3 and 30 cases of cervical invasive cancer. Double samples were collected from the same region. Fluorescence in situ hybridization was applied to detect the changes in the amplification of hTERC gene in 2 groups of specimens from the cervical biopsy.
 RESULTS: Differences in the positive expression rate of hTERC gene between the 2 groups of cervical lesions at all levels were not statistically significant (P>0.05).
 CONCLUSION: There is no significant difference in the positive rate of hTERC gene expression between the slices made by Van-clear and xylene. As an environmental-friend product, Van-Clear possesses certain value in detection of cervical hTERC gene by fluorescence in situ hybridization.

[Clinical analysis of cervical screening in 2329 pregnant women].

OBJECTIVE: To study the cytopathologic characteristics of cervical diseases in pregnant women and the outcomes of the postpartum women to provide evidence for prevention and treatment of cervical cancer. METHODS: This study was conducted among 2329 pregnant women undergoing routine gestational examinations between September, 2012 and September, 2013. The women with abnormal cytological findings by Thin-prep cytology test (TCT) were followed up and colposcopy and cervical biopsy were performed. The TCT results of these women were compared with those of 32 491 non-pregnant women in Zhongshan Cervical Cancer Mass Screening Program. RESULTS: Of the 2329 pregnant women, a total of 97 patients had abnormal TCT results (4.16%). Cervical biopsy were performed for 14 patients (14.43%), and 8 (57.14%) of them had evidence of cervical intraepithelial neoplasia (CIN) or cancer on biopsy. In the 32491 non-pregnant women in the mass screening program, 1383 (4.26%) women had abnormal TCT results and cervical biopsy were performed for 248 patients (17.93%), among whom 148 (59.68%) had evidence of CIN or cancer on biopsy. The rate of high-grade squamous intraepithelial lesion (HSIL) was significantly higher in non-pregnant women than in pregnant women (P=0.033), but the total rate of cytological abnormalities were comparable between them (P=0.911). The patients with CIN had regular examinations during pregnancy and postpartum follow-up showed no invasive carcinoma. CONCLUSION: Pregnancy is not a risk factor to accelerate the progress of cervical lesions, and most of the cervical lesions are relieved or show no progression in the postpartum women, suggesting the feasibility of follow-up during pregnancy and postpartum reevaluation for patients with CIN in pregnancy.

Cervical cancer screening and analysis of potential risk factors in 43,567 women in Zhongshan, China.

OBJECTIVE: The objective of this study was to establish a program model for use in wide-spread cervical cancer screening. METHODS: Cervical cancer screening was conducted in Zhongshan city in Guangdong province, China through a coordinated network of multiple institutes and hospitals. A total of 43,567 women, 35 to 59 years of age, were screened during regular gynecological examinations using the liquid-based ThinPrep cytology test (TCT). Patients who tested positive were recalled for further treatment. RESULTS: The TCT-positive rate was 3.17%, and 63.4% of these patients returned for follow-up. Pathology results were positive for 30.5% of the recalled women. Women who were younger than 50 years of age, urban dwelling, low-income, had a history of cervical disease, began having sex before 20 years of age, or had sex during menstruation, were at elevated risk for a positive TCT test. The recall rate was lower in women older than 50 years of age, urban dwelling, poorly educated, and who began having sex early. A higher recall rate was found in women 35 years of age and younger, urban dwelling, women who first had sex after 24 years of age, and women who had sex during menstruation. The positive pathology rate was higher in urban women 50 years of age and younger and women who tested positive for human papillomavirus. CONCLUSION: An effective model for large-scale cervical cancer screening was successfully established. These results suggest that improvements are needed in basic education regarding cervical cancer screening for young and poorly educated women. Improved outreach for follow-up is also necessary to effectively control cervical cancer.