Expression Of Survivin And Related Genes And Their Relationships With The Prognosis In Paediatric Osteosarcoma

【Objectives】Survivin is the most effective anti-apoptosis gene with the both functions of resistanting to cell apoptosis and regulation cell division recently,as well as contributing to cell proliferation and neoformative angiogenesis.There are some related genes such as Bcl-2,Ki67 and VEGF which have one of similar fuctions respectively.In this experiment,the expression of Survivin,VEGF,Bcl-2 and Ki67 were examined in children's osteosarcoma tissues by SP immunohistochemical technique,we explore whether these genes expressions are correlated with the onset,development,invasion and metastasis in human osteosarcoma and find values and relationships each other.And we try to explore whether Survivin expression is closely correlated with specific influence of the cell apoptosis,cell proliferation,neoformative angiogenesis,chemotherapy outcome and prognosis in osteosarcoma,which might be an important molecular prognostic marker in osteosarcoma.【Materials and Methods】This was a retrospective analysis of a consecutive series of 38 children with osteosarcoma from January 1990 to June 2008 in Children's hospital of Fudan University. Half were male and half were female,the average age was 9.5 years(range 4-14).Among 38 cases,25 cases in femur,9 cases in tibia,2 cases in fibula and 2 cases in humerus.According to the WHO classification system,35 cases of central medullary osteosarcoma;3 cases of surface osteosarcoma.A total of 34 osteosarcoma samples were obtained through biopsy and operation and had no case received chemotherapy or radiotherapy before treatment,28 cases had gained regular treatment and clinical and pathological documents were kept completely. Among 34 cases,one caseⅠgrade,11 casesⅡA grade,19 casesⅡB grade and 3 casesⅢgrade.One case was a 14 years boy and his father and father's younger brother died from osteosarcoma too.All samples were received random and blind analysis during SP immunohistochemistry study.All 28 cases were follow-up after operation,the survival patients were follow-up beyond 5 years.The expression of Survivin,VEGF,Bcl-2 and Ki67 were examined by SP immunohistochemistry in 34 cases of children's osteosarcoma and 15 cases of osteochondroma.We analyze the results of expression of survivin,VEGF,Bcl-2 and Ki67, find out the relationship of them and pathological grade,biological behavior in human osteosarcoma tissues.We explore relationship between the expression of them with tumor recurrence,distant metastasis and five year survival rate.In 28 cases with total follow-up informations,the expression of survivin were analyzed whether the expression were correlated with the chemotherapy response.The survival rates and survival curves were also compared between the negative and positive expressions of survivin gene.The possible factors which influenced prognosis in osteosarcoma were analyzed by COX multivariate analysis.COX multivariate regression identified the survivin expression levels as independent predictors of survival in osteosarcoma.SPSS12.0 software was used for statistics information.The averages between two groups were analyzed by T-test while the two sample rates were compared by Chi-square test.And spearman correlation coefficient was also used,the survival curve was analyzed by Kaplan-Meier method.We also used COX proportional hazard regression model multivariate analysis.Values of P＜0.05 were considered to indicate statistical significance.【Results】In all the 34 cases of osteosarcoma,the positive rate of Survivin was 67.6%(23/34),as well as none was detected in 15 cases of osteochondroma(P＜0.01).The positive survivin expression was correlated with Enneking grade but not correlated with the gender,age, primary location,tumor diameter and pathological grade.The difference between the I-IIA group andⅡB-Ⅲgroup was significant(P＜0.01).The positive rate of Survivin in distant lung metastasis group was 86.7%(13/15);While the positive rate of Survivin in the 13 cases of no metastasis was 46.1%(6/13).The five year survival rate of the positive Survivin group was 21.1%(4/19) much lower than that[66.7%(6/9)]of the negative survivin group.The differences were all statistically significant(P＜0.05).In all the 34 cases of osteosarcoma,the positive rate of VEGF was 73.5%(25/34),as well as none was detected in 15 cases of osteochondroma(P＜0.01).The positive VEGF expression was correlated with Enneking grade but not correlated with the gender,age,primary location, tumor diameter and pathological grade.The difference between theⅠ-ⅡA group andⅡB-Ⅲgroup was significant(P＜0.01).The VEGF expression much higher when Enneking grade higher.The positive rate of VEGF in distant lung metastasis group was 86.7%(13/15);While the positive rate of VEGF in the 13 cases of no metastasis was 69.2%(9/13).The five year survival rate of the positive VEGF group was 27.3%(6/22) much lower than that[66.7%(4/6)] of the negative VEGF group.But the differences were no obviously statistically significant(P＞0.05).In this study,the VEGF positive expression rate of the positive survivin group was 87.0%(20/23) much higher that[45.5%(5/11)]of the negative survivin group. Which showed statistically significant(X~2=6.33,P＜0.05).The spearman correlation coefficient indicated the expression of survivin was positively correlated with VEGF in the cases of osteosarcoma(r=0.652,P＜0.05).In all the 34 cases of osteosarcoma,the positive rate of Bcl-2 was 41.2%(14/34),as well as none was detected in 15 cases of osteochondroma(P＜0.01).The positive Bcl-2 expression was correlated with Enneking grade but not correlated with the gender,age,primary location, tumor diameter and pathological grade.The difference between theⅠ-ⅡA group andⅡB-Ⅲgroup was significant(P＜0.05).The Bcl-2 expression much higher when Enneking grade higher.The positive rate of Bcl-2 in distant lung metastasis group was 60.0%(9/15);While the positive rate of Bcl-2 in the 13 cases of no metastasis was 23.1%(3/13).The five year survival rate of the positive Bcl-2 group was 33.3%(4/12) lower than that[37.5%(6/16)]of the negative Bcl-2 group.But the differences were no obviously statistically significant(P＞0.05). The result was similar to the other literatures report.In this study,the Bcl-2 positive expression rate of the positive survivin group was 56.5%(13/23) much higher that[9.1% (1/11)]of the negative survivin group.Which showed statistically significant(X~2=7.88, P＜0.01).The spearman correlation coefficient indicated the expression of survivin was positively correlated with Bcl-2 in the cases of osteosarcoma(P＜0.01).The positive Ki67 expression was not correlated with the gender,age,primary location, tumor diameter,pathological grade and Enneking grade(P＞0.05).The Ki67-LI expression of the positive survivin group was(38.4±20.3)%,the positive rate was 69.6%(16/23);the negative survivin group was(17.5±13.8)%,the positive rate was 27.3%(3/11).The difference was also statistically significant(P＜0.05).The chemosensitivity rate of positive survivin group was 38.1%(8/21),significantly lower than that of the negative survivin group,which was 71.4%(5/7);The distant lung metastasis rate of positive survivin group post-chemotherapy was 71.4%(5/7),While the negative group was 47.6%(10/21),there was obviously different,but the differences were no obviously statistically significant(P＞0.05) because of small samples.The five year survival rate of positive survivin group was 21.1%(4/19),significantly lower than that of the negative survivin group,which was 66.7%(6/9);The survival curve was analyzed by Kaplan-Meier method,the survival curves between the two groups showed statistically significant(X~2=8.900,P=0.003＜0.01).The possible factors which influenced prognosis in osteosarcoma were analyzed by COX multivariate analysis.COX multivariate regression identified the survivin expression levels and Enneking grade as independent predictors of survival in osteosarcoma. 【Conclusions】1.The survivin protein is specially expressed in osteosarcoma.The expression is closely correlated with relapse and metastasis in osteosarcoma,which indicates that it may play an important role in the onset and development of osteosarcoma.Survivin gene might be a new molecular diagnostic marker in osteosarcoma.The positive survivin expression was correlated with Enneking grade but not correlated with the gender,age,primary location,tumor diameter and pathological grade.COX multivariate regression identified the survivin expression levels and Enneking grade as independent predictors of survival in osteosarcoma.2.The expression of survivin is positively correlated with VEGF,Bcl-2,Ki67 in the cases of osteosarcoma,which indicated Survivin expression is closely correlated with specific influence of the cell apoptosis,cell proliferation,neoformative angiogenesis,chemotherapy outcome and prognosis in osteosarcoma,which might be an important molecular prognostic marker in osteosarcoma.3.The expression of survivin reduces the chemosensitivity.Survivin levels in both initial biopsy and post-chemotherapy samples are useful prognostic indicators.