| BACKGROUND:Cervical cancer is one of the most common neoplasm in women,after breast cancer.The incidence of cervical cancer in China is gradually increased over the past decade,and patients tend to be more younger.The mortality of cervical cancer has been decreased significantly in developed countries,as the depth understanding of history of cervical cancer risk factors,appropriate screening techniques and comprehensive physical examination.However,the incidence and mortality of cervical cancer remains higher in most developing countries.In China,the number of new cases of cervical cancer are more than 130000,which killed are at least 20000 to 30000.Cervical intraepithelial neoplasia lesion is precancerous lesion,which is closely related with the the occurrence of cervical cancer.Therefore,the research on these molecular markers which may be associated with the formation and development of cervical cancer will not only help elucidate the pathogenesis of the cervical cancer,but also be conducive to the early diagnosis,treatment and prognosis estimation.PBK/TOPK(PDZ-binding kinase/T-LAK cell-originated protein kinase)is a novel protein kinase,which was early found in lymphokine-activated killer T cells(T-LAK),and closely related to the occurrence and development in a variety of malignancies.PBK/TOPK highly expressed in many kinds of malignant tumors,but is difficult to be detected in normal tissues.At present,there is no report to estimate whether PBK/TOPK expression is involved in the occurrence and development of cervical cancer or not,so further studies are needed.P16,a novel tumor suppressor gene,is prone to mutation and deletion,which is involved with the occurrence and development of a variety of tumors.Ki-67 is a common marker of cell proliferation assays,and widely used in clinical and pathological diagnosis.P53 is a tumor suppressor gene,whose deletion or mutation is closely related to the occurrence and development of types of tumors.Currently,some studies reported that P16,Ki-67 and P53 are involved in the pathogenesis of cervical cancer.However,the relationships of PBK/TOPK with P16,Ki-67 and P53 in cervical cancer is unclear,so we carried out the study.OBJECTIVES:1.To investigate the expression and prognostic significance of PBK/TOPK in cervical cancer and cervical intraepithelial neoplasia.2.To investigate the correlation and prognostic significance of PBK/TOPK expression with P16,Ki-67 and P53 expression in cervical correlation.3.To investigate the correlation and prognostic significance of P16 expression with Ki-67 and P53 expression in cervical cancer.4.To investigate the expression and prognostic significance of Ki-67 and P53 in cervical lesions.Materials and Methods:1.The expression and prognostic significance of PBK/TOPK in cervical cancer and cervical intraepithelial neoplasia PBK/TOPK expression was detected in 28 cases of CIN 1,62 cases of high-grade intraepithelial neoplasia and 80 cases of cervical cancer by SP immunohistochemical method.ImageproPlus6.0(IPP)image analysis software testing was used to quantificationally assess the positive unit(PU value)of PBK/TOPK expression,and data were performed statistical analysis and postoperative survival analysis by using semi-quantitative analysis combined with relevant clinical data and follow-up.2.The correlation and prognostic significance of PBK/TOPK with P16,Ki-67 and P53 in cervical cancerImmunohistochemistry was used to detect the expression of PBK/TOPK,P16,Ki-67 and P53 in 80 cases of cervical cancer.Using quantitative and semi-quantitative method,immunohistochemical results were analyzed with clinicopathological data and postoperative follow-up data to explore the correlation of PBK/TOPK with P16,Ki-67 and P53 in cervical cancer.3.The correlation and prognostic significance of P16 with Ki-67 and P53 in cervical cancerImmunohistochemistry was used to detect the expression of P16 with Ki-67 and P53 in 80 cases of cervical cancer.Using quantitative and semi-quantitative method,immunohistochemical results were analyzed with clinicopathological data and postoperative follow-up data to explore the correlation of P16 with Ki-67 and P53 in cervical cancer.4.The expression and prognostic significance of Ki-67 and P53 in cervical lesionsImmunohistochemical method was used to detect the expression of Ki-67 and P53 in 7 cases of cervicitis,38 cases of CIN I,79 cases of high-grade intraepithelial neoplasia and 91 cases of cervical cancer.The relationship and prognostic significance between Ki-67 and P53 in cervical lesions were also investigated.The reference standard of semi-quantitative determination:Combining the color intensity and the percentage of cells,the expression of PBK/TOPK,P16,Ki-67 and P53 were assessed.The color intensity scores ranged from 0 to 3,0 point:colorless;1 point:faint yellow;2 points:yellow;3 points:brown.The positive cell percentage score ranged from 1 to 4 points,1 point:0-10%;2 points:11%-24;3:25%-50%;4 minutes:>50%.When total scores were greater than or equal to 6 points,we defined them as high expression.On the other hand,the total scores will be defined as low expression.The reference standard of quantitative determination:On the premise that there were no false positives or false negatives,those with dark brown particles in the nuclei or the cytoplasm were regarded as positive protein.The positive value(PU)was measured by ImagePro Plus.The procedure is as followed:10 objectives with positive protein expression taken at 40 X objective lensthe were converted into a gray scale image.Interactive method was used to measure the gray level(G?)of each positive cell.Background gray level(G?)was measured in the same field.According to the formula below,positive unit(PU)of each positive cell was calculated.The average value of the PU of 200 positive cells was regarded as the PU of the specimen.PU =|G?-G?|/Gmax×100Gmax=2565.Statistical AnalysisStatistical analysis was performed using SPSS 17.0 software.The comparison of two sample means was performed by independent-sample t-tests,and the comparison of multiple sample means was performed using one-way ANOVA test.The correlation analysis was assessed by Spearman rank correlation.Survival analysis was estimated by Kaplan-Meier method with log-rank test,and Cox's proportional hazards model was for multivariate analysis.Statistical significance was defined as the 2-sided p-value lower than 0.05.Results:1.The expression and prognostic significance of PBK/TOPK in cervical cancer and cervical intraepithelial neoplasiaThe quantitative test results of positive unit(PU)showed that PBK/TOPK expression was significantly higher in cervical cancer tissues than that in the high-grade intraepithelial neoplasia and CIN I tissues,with a significant differences(P=0.000,P=0.000).PBK/TOPK expression was significantly higher in the high-grade intraepithelial neoplasia tissues than CIN I tissues,with a significant difference(P=0.000).PBK/TOPK expression was associated with histological type,differentiation degree,lymph node metastasis,vagina and cervix involvement,tumor stage and tumor size(P<0.05),whereas no significant correlation with age was found(P>0.05)in cervical tissues.The 3-year and 5-year overall survival rates in patients with high PBK/TOPK expression were 88.3%,74.1%,and were 100%,75.0%in other patients with low PBK/TOPK expression,no significant statistical difference was found(P>0.05).Univariate Kaplan-Meier and Cox proportional hazards regression model analysis showed that lymph node metastasis rather than PBK/TOPK expression,age,differentiation degree,vaginal and cervical involvement,tumor stage and tumor size was correlated with patients' prognosis.2.The correlation and prognostic significance of PBK/TOPK with P16,Ki-67 and P53 in cervical cancerThe quantitative detection results showed that PBK/TOPK was positively correlated with P16 in cervical cancer(r=0.375,P=0.000).Meanwhile,semi-quantitative analysis showed that the high-expression rates of PBK/TOPK and P16 in cervical cancer rate were 75.0%,81.3%,respectively,with a significant difference(P=0.000).Otherwise,there was a positive correlation between PBK/TOPK expression and P16 expression(r=0.684,P=0.000).Univariate Kaplan-Meier survival analysis showed that there was a significant statistical difference in PBK/TOPK low/P 16 low group,PBK/TOPK low/P 16 high group,PBK/TOPK High/P16 low group and PBK/TOPK high/P16 high group(P=0.000),and patients in PBK/TOPK high/P 16 low group had poorer prognosis.Quantitative analysis showed that PBK/TOPK expression was positively correlated with Ki-67 expression(r=0.407,P=0.000).semi-quantitative analysis showed that the high-expression rates of PBK/TOPK and P16 in cervical cancer rate were 75.0%,72.5%,respectively,without a significant difference(?2=0.129,P=0.858),however,there was a clear positive correlation between PBK/TOPK and P16 expression(r=0.555,P=0.000).Kaplan-Meier survival analysis showed that there wasn't a significant statistical difference in PBK/TOPK low/Ki-67 low group,PBK/TOPK low/Ki-67 high group,PBK/TOPK high/Ki-67 low group and PBK/TOPK high/Ki-67group(x2=3.178,P=0.365).Quantitative analysis showed that PBK/TOPK was positively associated withP53 in cervical cancer(r=0.351,P1=0.001).Semi-quantitative analysis showed that the high-expression rate of PBK/TOPK and P53 in cervical cancer were 75.0%,42.5%,respectively,and the difference was statistically significant(?2=17.434,P=0.000).The results also showed there is a clear positive correlation between PBK/TOPK expression and P53 expression(r=0.38,P=0.001).Univariate Kaplan-Meier survival analysis showed that there is no significant statistical difference between the four groups of PBK/TOPK low/P53 low,PBK/TOPK low/P53 high,PBK/TOPK high/low P53 and PBK/TOPK high/P53 high(?2=6.612,P=0.085).3.The correlation and prognostic significance of P16 expression with Ki-67 and P53 expression in cervical cancerQuantitative analysis showed that P16 expression was positively correlated with Ki-67 expression in cervical cancer(r=0.487,P=0.000).Semi-quantitative analysis showed the high-expression rates of P16 and Ki-67 in cervical cancer were 81.3%,72.5%,respectively,without a statistical significant difference(?2=1.723,P=0.26).The results also showed there is a clear positive correlation between P16 expression and Ki-67 expression(r=0.637,P=0.000).Kaplan-Meier survival analysis showed that P16 expression is not significantly correlated with patients'prognosis(P>0.05),while there was a statistically significant difference in P16 low/Ki-67 low group,P16 low/Ki-67 high group,P16 High/Ki-67 low group,P16 high/Ki-67 high group(?2=38.67,P=0.000),and patients in P16 low/Ki-67group had poorer prognosis.Quantitative analysis showed that P16 expression was positively related to P53 expression in cervical cancer(r=0.482,P=0.000).Semi-quantitative analysis showed that the high-expression rates of P16 and P53 in cervical cancer were shown 81.3%to 42.5%,respectively,with a significant difference(?2=10.620,P=0.002).Also,the results indicated there was a positive correlation between P16 expression and P53 expression(r=0.413,P=0.000).Univariate Kaplan-Meier survival analysis showed that there was a significant difference in the four groups of P16 low/P53 low,P16 low/P53 high,P16 High/P53 low,P16 High/P53 high(?2= 37.708,P=0.000),and patients with P16 low/P53 high had poorer prognosis.4.The expression and prognostic significance of Ki-67 and P53 in cervical lesionsQuantitative test results showed that Ki-67 expression was higher in high-grade intraepithelial neoplasia and cervical carcinoma than that in CIN I and cervicitis tissues,and the differences were statistically significant(P<0.05).P53 expression in the high-grade intraepithelial neoplasia and cervical carcinoma tissues was significantly higher than CIN I and cervicitis tissues,with a significant difference(P<0.05).Quantitative test results showed Ki-67 expression positively correlated with P53 expression in cervical carcinoma(r=0.577,P=0.000).Meanwhile,semi-quantitative analysis showed the high-expression rates of Ki-67 and P53 in cervical cancer were 75.8%,41.8%,respectively,with a significant difference(?2=21.795,P=0.000).The results also showed there was a significant positive correlation between Ki-67expression and P53 expression(r=0.322,P=0.002).Kaplan-Meier survival analysis showed that Ki-67 and P53 expression was not significantly correlated with patients' prognosis(?2=0.499,P=0.480;?2=3.288,P=0.07).Combining Ki-67 expression and P53 expression,there wasn't a significant difference in the four groups of Ki-67 low/P53 Low,Ki-67 low/P53 high,Ki-67 high/P53 low and Ki-67 high/P53 high(?2=3.35,P=0.341).Conclusions:1.PBK/TOPK expression is closely related to the progression of cervical intraepithelial neoplasia and the occurrence and development of cervical cancer.2.PBK/TOPK expression was positively correlated with the expression of P16,Ki-67 and P53 in cervical cancer,and patients with.PBK/TOPK high/P16 low have poorer prognosis.3.P16 expression was positively correlated with the expression of Ki-67 and P53 in cervical cancer,and patients with P16 low/Ki-67 high or P16 low/p53 high have poorer prognosis.4.Ki-67 and P53 were involved in the progression of cervical intraepithelial neoplasia and the occurrence and development of cervical cancer,and there is a positive correlation in their expression. |
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