The Test Threshold And Prognosis Significance Study Of Ki-67 And P53 Protein In Lung Cancer

Since the mid-20th century, the incidence rates of lung cancer show a rising trend in most countries and the constant increasingly year by year, its incidence and mortality of various malignant tumors accounted for the top. Ki-67 is currently the most widely used marker of proliferative cells, It is very important that helping the diagnosis, prognosis and guiding treatment. About Ki-67 and histological type, differentiation, pTNM stage, lymph node status, age and smoking relations of lung cancer have different views, and study qualitative analysis at different angle. There are many different opinions in term of the connection of Ki-67 and lung cancer prognosis, so, Ki-67 and lung cancer prognosis has been the research focus. Therefore, the in-depth study Ki-67 and prognosis of lung cancer has important practical significance. p53 as tumor suppressor gene was value by the people, p53 mutations in lung cancer is being prevalence in the process. p53 gene mutation or p53 protein expression has relevance of biological behavior and clinical pathological features, is still controversial. Reported that the expression of p53 and Ki67 was positively correlated, but there was a negative correlation between the two reported in the literature. Well, how is the relation between Ki-67 and p53 in the end? Whether will be a new discovery about the joint detection of Ki-67 and p53 in the prognosis for lung cancer? In the end result, it would need more clinical studies further confirmed. Moreover, in the past for lung cancer Ki-67 and p53 expression as qualitative observations how is quantitative expression features based on quantitative changes?OBJECTIVE AND SIGNIFICANCEThe topic was to evaluate the quantitative expression of Ki-67 and p53 protein in lung cancer tissue, normal adult lung tissue and benign diseases lung tissue of adults, from protein level revealed Ki-67 and p53 the quantitative characteristics of lung cancer,and study Ki-67 and p53 protein in lung cancer diagnosis and differential diagnosis of clinical value, and indicates the potential role of the combination of Ki-67 and p53 in lung cancer prognosis.METERLAILS AND METHODSⅠ,The quantitative analysis of Ki-67 and p53 expression in lung cancerExperimental materials and grouping:Tissues from the autopsy biopsy specimens of Nan fang Hospital Pathology Department between 2000-2009. Patients did not receive radiotherapy or chemotherapy before operation. Samples is as follows: 18 cases of normal adult lungs,50 cases of adult group with benign lung diseases,161 cases of lung cancer patients, according to 1999 WHO/IASLC histopathology grade classification of lung cancer, lung cancer will be further divided into:73 cases of squamous cell carcinoma,59 cases of adenocarcinoma,14 cases of adenosquamous carcinoma,5 cases of large cell carcinoma,10 cases of small cell carcinoma.104 cases lung cancer with lymph node metastasis,57 cases lung cancer with no lymph node metastasis,43 cases lung cancer with distant metastasis,118 cases lung cancer with no distant metastasis,48 cases of central lung cancer,113 cases of peripheral lung cancer. Of 34 patients of TNM stageⅠ,29 cases of TNMⅡ,55 cases of TNMⅢ,43 cases of TNMⅣ.Experimental Methods:Detected the expression of Ki-67 and p53 protein in lung tissues of different types by immunohistochemical, application of Leica Q500 MC image analysis system test Ki-67 and p53 protein-positive units(PU) value,than analyze the changings of Ki-67 and p53 expression intensity between different types of lung tissues,and analyze the relations between clinical and pathological features of lung cancer.Statistical analysis:SPSS 13.0 statistical analysis software application for processing. Comparison of samples averages were used to One-way ANOVA; multiple comparisons between groups use Dennett's T3 test; sex, smoking history, lung dust conditions, tumor gross type, tumor differentiation degree, lymph node metastasis, distant metastasis and comparison of TNM staging of lung cancer was used to two sample t test.Ⅱ,The diagnosis analysis of Ki-67 and p53 in lung cancerExperimental materials and grouping:See 1(The quantitative analysis of Ki-67 and p53 expression in lung cancer),and the difference between chapter 1 and chapter 2 is that the lung cancer tissues is 168 cases in this chapter,for other pathological types of lung cancer (for example:carcinoid, spindle cell carcinoma, etc).Experimental Methods:application of immunohistochemical techniques and Leica Q500 MC image analysis system detect the expression of Ki-67 and p53 protein in lung cancer tissue, normal adult lung tissue, benign diseases lung tissue,than combinate with diagnostic test assessment method to determine the diagnostic cut off value of Ki-67 and p53,reveals the diagnosis and differential diagnosis of lung cancer in a supporting role. Statistical analysis:SPSS13.0 statistical analysis software application for processing. Application of diagnostic test tables from different truncation values obtained and diagnostic parameters, combined with receiver operation characteristic curve(ROC curve)analysis Ki-67 and p53 protein cut off value.Ⅲ,The survival analysis of Ki-67 and p53 in lung cancer prognosisExperimental Materials:This study is retrospective,collected 161 cases from the South Hospital in 2000-2005 which meet diagnostic criteria and inclusion criteria:including 132 patients of detailed and complete clinical follow-up data, average age is 56.77±9.78(34-73years);including 104 males average age is 57.23±9.69(34～73years); including 28 females average age is 55.07±10.10(35～73years);the remaining 29 cases were lost, as the censored data.Experimental Methods:According to immunohistochemical expression of Ki-67 and p53 in lung cancer tissue,we collected clinical and follow-up data of lung cancer and divided the lung cancer patients into four group by semi-quantitative method:A group(Ki-67 high expression/p53 high expression),B group(Ki-67 high expression/p53 low expression),C group(Ki-67 low expression/p53 high expression),D group(Ki-67 low expression/p53 low expression),to discuss the difference between the survival time of four groups; and using Univariate analysis and COX regression model revealed the influence on the survival time of patients.Statistical analysis:SPSS13.0 statistical analysis software application for processing. Kaplan-Meier method used in the calculation of median survival time, significance test by Log rank test, R×C data x2-test was used to compare 1-year to 5-year survival rate between groups,the COX regression analysis of prognostic factors. Survival time was calculated from the date of diagnosis to death or last follow-up time.Many samples used to compare the analysis of variance; test of homogeneity of variance among the samples to know the overall homogeneity of variance, multiple comparisons between groups use LSD test; if missing the overall variance between groups, multiple comparisons use Dunnett's T3 test.RESULTSⅠ,The quantitative analysis of Ki-67 and p53 expression in lung cancer1. Ki-67 PU value of normal adult lung tissue and benign disease lung tissue is less than Ki-67 PU values of squamous cell carcinoma,adenocarcinoma, adenosquamous carcinoma and small cell lung carcinoma(P=0.000);Ki-67 PU value of normal adult lung tissue and benign disease lung is less than Ki-67 PU values of large cell carcinoma(P=0.028);Ki-67 PU value of normal adult lung tissue is basically the same with Ki-67 PU values of benign disease lung tissue(P=0.991); Ki-67 PU values of squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma,large cell carcinoma and small cell carcinoma of the lung cancer are similar (P> 0.05). p53 PU value of normal adult lung tissue and benign disease lung tissue is less than p53 PU values of squamous cell carcinoma,adenocarcinoma(P= 0.000); p53 PU values of normal adult lung tissue is basically same to p53 PU value of benign disease lung tissue (P=0.995);squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma,large cell carcinoma is basically same to p53 PU value of small cell carcinoma of the lung cancer(P> 0.05).2. Ki-67 and p53 PU values of lung cancer cell nuclear is indifferent with sex (P=0.603; P=0.317), smoking history (P=0.110; P=0.328), pneumoconiosis cases (P=0.301; P=0.070), tumor gross type (P=0.366; P=0.343), degree of differentiation of lung cancer (squamous cell carcinoma:P=0.605; P=0.066; adenocarcinoma: P=0.879; P=0.867), lymph node metastasis (P=0.658;P=0.797), distant metastasis (P=0.550; P=0.714) and TNM stage (P=0.420; P=0.737).3. From the view of semi-quantitative showed that:There was no correlation between Ki-67 expression and p53 expression(r=0.119, P=0.132). Ki-67 and p53 consistency analysis showed that:Kappa=0.11, consistency is poor.Ⅱ,The diagnosis analysis of Ki-67 and p53 in lung cancer1. Application of receiver operation characteristic curve(ROC curve) analyzed Ki-67 protein truncation value, when you select x+4s as Ki-67 normal cutoff value of lung tissue, this is 16.09, the detection sensitivity, specificity, and accuracy are at a good level.And the ROC value is 0.96.Furthermore,the standardization diagnostic tests have sensitivity (Se) was 91.07%, specificity (Sp) was 100.00%, standardized positive predictive value(SPPV) was 100.00%, standardization negative predictive value (SNPV) was 91.80% and the standardized accuracy (SAc) was 95.54%, standard error diagnosis rate (SWDR) was 4.46%.2. Application of ROC curve analyze cut-off value of p53 protein,when you select x+4s as p53 normal cut off value of lung tissue p53, that is 17.20, the sensitivity, specificity, accuracy are at a good level. At this time,ROC value is 0.62.Furthermore,standardization diagnostic tests have sensitivity (Se) was 55.95%, specificity (Sp) was 97.06%, standardized positive predictive value(SPPV) was 95.05%, standardization negative predictive value (SNPV) was 68.79%, standard accuracy (SAc) was 76.52%, standard error diagnosis rate (SWDR) was 23.48%.3. Ki-67 and p53 series experiments sensitivity was 50.00%, specificity was 100%; Ki-67 and p53 parallel experiments sensitivity was 93.45%, specificity was 100%.Ⅲ,The survival analysis of Ki-67 and p53 in lung cancer prognosis1. Clinical and pathological features of lung cancer is affect on the prognosis of patients with single-factor analysis showed that:lymph node metastasis, TNM stage, Ki-67 and p53 combined detection of these factors affect the prognosis of survival time, the differences were statistically significant (P<0.05). Other factors have no significant effect with the prognosis survival time of patients, the differences were not statistically significant (P> 0.05).2. The correlation analysis between positive rate of Ki-67 and survival time of patients of lung cancer showed that:r=-0.196, P=0.024,the correlation was significant, but not closely correlated. The correlation analysis between positive rate of p53 and survival time of patients of lung cancer showed that:r=0.004, P=0.967, there was no correlation.3. Ki-67 and p53 combined to detect lung cancer A group(Ki-67 high/p53 high), B group(Ki-67 high/p53 low), C group(Ki-67 low/p53 high), D group(Ki-67 low/p53 low)changes in survival time of patients:A group of median survival time was 24 months,1,2,3,4 and 5-year survival rates were 90.0%,60.0%,40.0%,30.0% and 26.7%; B group of median survival time was 16 months,1,2,3,4 and 5-year survival rates were 74.4%,44.2%,20.9%,14.0% and 11.6%; C group of median survival time was 22 months,1,2,3,4 and 5-year survival rates were 70.0%,50.0%,40.0%,20.0% and 20.0%; D group of median survival time was 24 month, 1,2,3,4 and 5-year survival rates were 89.7%,69.2%,48.7%,46.2% and 35.9%. A, B, C, D four groups was statistically significant difference in survival time (P= 0.023); of which, survival time of A and D group are more than B group, the difference was statistically significant (P=0.033 and P=0.006).4-year survival rate of D group was significantly better than A, B, C three groups,4-year survival rate of B group is lowest, the difference was statistically significant (P=0.010). Among the four groups, the difference of 1,2,3 and 5-year survival rate was not statistically significant (P values were 0.091,0.127,0.063 and 0.072).4. Ki-67 and p53 combined to detect non-small cell lung cancer A group (Ki-67 high/p53 high), B group (Ki-67 high/p53 low), C group (Ki-67 low/p53 high), D group (Ki-67 low/p53 low)changes in survivalb time of patients:A group of median survival time was 30 months,1,2,3,4 and 5-year survival rates were 96.2%,61.5%,46.2%,34.6% and 30.8%; B group, the median survival time was 16 months, 1,2,3,4 and 5-year survival rates were 77.5%,45.0%,22.5%,15.0% and 12.5%; C group the median survival time was 24 months,1,2,3,4 and 5-year survival rates were 73.7%,52.6%,42.1%,21.1% and 21.1%; D group the median survival time was 34 months,1,2,3,4 and 5-year survival rates were 92.1%,71.1%,52.6%,50.0% and 39.5%. A, B, C, D four groups was statistically significant difference in survival time (P=0.024); of which, survival time of A group and D group more than B group, the difference was statistically significant (P=0.019 and P=0.011). D group 1,3,4, and 5-year survival rate was significantly better than A, B, C three groups, B group 1,3,4 and 5 year survival rate in the lowest, the difference was statistically significant (P values were 0.049,0.044,0.007,0.048). A, B, C, D 2-year survival rate among the four groups showed no significant difference (P=0.123).5. There are 46 cases lung cancer patients without lymph node metastasis, the median survival time was 34 months, there are 86 cases lung cancer patients with lymph node metastasis, the median survival time was 21 months, the survival time without lymph node metastasis is longer than those with lymph node metastasis. The difference was statistically significant (P=0.005). TNMⅠ,Ⅱ,Ⅲ,Ⅳstage of the patients were 26 cases,24 cases,44 cases and 38 cases, the median survival time was 60 months,18 months,20 months and 24 months, the difference was statistically significant (P=0.001).6. (1)Survival time of patients will be divided into six groups:<12m,12-23m, 24～35m,36～47m,48～59m,≥60m, the comparison of the PU values of six groups of Ki-67 and p53 were not statistically significant (P> 0.05).(2) There is comparison of the PU value of Ki-67 and p53 between A group(Ki-67 high/p53 high), B group(Ki-67 high/p53 low), C group(Ki-67 low/p53 high), D group(Ki-67 low/p53 low):overall, four groups have statistically significant difference(F=12.071, P=0.000; F=68.137,P=0.000).7. Cox regression analysis about gender, age, smoking history, lung dust conditions, gross type, histological type, differentiation degree, lymph node metastasis, distant metastasis, TNM stage, the percentage of positive cells of Ki-67 and p53, independent testing of Ki-67 and p53, joint detection of Ki-67 and p53. It is show that TNM stage was independent prognostic factor which affects survival (P=0.030), suggest that:the greater the TNM staging, the shorter survival time.CONCLUSIONS1. From the quantitative view confirmed that:Ki-67 and p53 expression in lung tissues is higher than in normal adult lung tissue and benign disease lung tissue; there was no difference between normal adult lung tissue and benign disease lung tissues; there was no difference between all histological types of lung cancer; Ki-67 and p53 PU values of lung cancer cell nuclear is indifferent with sex, smoking history, pneumoconiosis cases, tumor gross type, degree of differentiation of lung cancer, lymph node metastasis, distant metastasis and TNM stage. There was no correlation between Ki-67 expression and p53 expression; Ki-67 and p53 consistency is poor.2. When the cutoff value of Ki-67 is 16.09,the lung cancer sensitivity was 91.07%, specificity was 100.00%;when the cutoff value of p53 is 17.20,the lung cancer sensitivity was 52.38%, specificity was 100.00%; Ki-67 and p53 parallel experiments sensitivity was 93.45%, specificity was 100%,its value in diagnosis is highest.3. The joint detection of Ki-67 and p53 is better than independent predictors of lung cancer.1～5-year survival rate were:Ki-67 low/p53 low group (34 months)> Ki-67 high/p53 high group (24 months)> Ki-67 low/p53 high group (22 months)> Ki-67 high/p53 low group (16 months).