Expression Of CCNB2 And Its Correlation With The Clinicopathologic Features And Prognosis Of NSCLC

BackgroundLung cancer is the leading cause of cancer related death worldwide in both men and women. Based on 2015 Cancer Statistics, a total of estimated 221,200 new lung cancer patients and 158,040 lung cancer patient deaths occur in the America in 2015. Non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) are main subtypes of lung cancer, and NSCLC represents 80-85% of all lung cancer cases. Despite developments of early diagnosis and novel systemic treatment, NSCLC is generally diagnosed at an advanced stage, and the overall 5-year overall survival for NSCLC is 15%. The failure of NSCLC therapy and poor prognosis of the disease are mainly attributed to tumor invasion, metastasis, and recurrence. Therefore, novel biomarkers, which predict malignant status and poor prognosis of NSCLC patients, are urgently needed to identify high-risk patients and guide treatment strategies.Cyclin B2 (CCNB2) is a member of cyclin family proteins, which generally triggers the progression of G2/M transition through activating CDK1 kinase. Recently, more and more evidences suggested that CCNB2 expression was increased in a variety of human cancers, such as adrenocortical carcinoma, breast carcinoma, colorectal adenocarcinoma, pituitary adenoma and gastric cancer. Moreover, serum circulating CCNB2 mRNA expression has been found increased in cancer patients and associated with cancer stage and metastasis status. The significance of CCNB2 protein expression in lung cancer is still unclear. So we further explore the relationship between the expression of CCNB2 and its correlation with the clinicopathologic features and prognosis of NSCLC. Our research objectives include the following aspects:1. To examine the differences of expression of CCNB2 in non-small cell lung cancer tissue and normal tissue;2. To clarify the relationship between CCNB2 expression and the clinico-pathological features of non-small cell lung cancer;3.To identify the relationship between CCNB2 expression and survival of the non-small cell lung cancer patients.Material and Method1. Sample collectionA total of 20 pairs of freshly-frozen non-small cell lung cancer tissues samples and normal lung tissue samples were collected from Nanfang Hospital and Fifth People’s Hospital of Fuyang. All fresh samples were immediately preserved in liquid nitrogen.186 paraffin-embedded non-small cell lung cancer specimens and 31 paraffin-embedded normal lung tissue specimens were retrieved from Nanfang Hospital, Fuyang Second People’s Hospital, Huanggang Central Hospital, Fifth People’s Hospital of Fuyang and Fuyang Tumor Hospital, of which the patients were diagnosed between January 1,2008 and June 30.2014. We performed real-time PCR to measure the expression of CCNB2 mRNA transcripts in fresh NSCLC tissues and paired adjacent normal lung tissues. We detected the expression levels of CCNB2 protein in archived paraffin-embedded NSCLC samples and normal lung samples using immunohistochemical staining.2. Statistical analysisAll data were analyzed for statistical significance using SPSS 13.0 software. The Wilcoxon Signed Rank test was used to examine the differences of CCNB2 mRNA expression between non-small cell lung tissues and paired adjacent normal lung tissues. The chisquare test was used to examine the differences of CCNB2 protein expression between normal and cancer tissues. The chi-square test was applied to the examination of relationship between CCNB2 expression and clinicopathological characteristics. Survival analysis was performed using Kaplan-Meier method and Coxmultivariate proportional hazards model.Results1. CCNB2 mRNA expression in adjacent tissues and cancer tissuesBased on Lu et al.’s microarray data (GSE19804), CCNB2 was found overexpressed in sixty NSCLC tissues compared with paired adjacent normal lung tissues.In order to verify the status of CCNB2 in NSCLC, we performed real-time PCR to measure the expression of CCNB2 mRNA transcripts in twenty fresh NSCLC tissues and paired adjacent normal lung tissues. This expression pattern was similar to the microarray data. Compared with paired adjacent normal lung tissues, the levels of CCNB2 mRNA was overexpressed with an average increase of 3.02-fold (P < 0.001).We detected the expression levels of CCNB2 protein in 186 archived paraffin-embedded NSCLC samples and 31 normal lung samples using immunohistochemical staining. We observed that CCNB2 protein was overexpressed in 62.4%(116/186) of NSCLC samples. In comparison, only 29.0% of normal lung tissues had overexpressed CCNB2 protein, significantly lower than that in the NSCLC tissues (P= 0.002).2. CCNB2 protein expression and clinicopathological features of NSCLCWe further analyzed the association between CCNB2 expression and clinicopathological characteristics of NSCLC patients. CCNB2 protein overexpression was significantly associated with differentiated degree (High vs. middle; P< 0.001 and High vs. Low; P< 0.001), tumor size (T1-T2 vs. T3-T4; P= 0.007), lymph node metastasis (N0-N1 vs. N2-N3; P< 0.001), distant metastasis (M0 vs. M1; P= 0.002), and clinical stage; P(Ⅰ-Ⅱ vs. Ⅲ-Ⅳ; P< 0.001). However, there was no significant correlation between CCNB2 expression and gender Female vs. Male; P= 0.460), age (<50 vs.50; P= 0.222), smoking (No vs. Yes; P= 0.785) and pathology classification (Squamous cell carcinoma vs. Adenocarcinoma; P= 0.759).3. CCNB2 overexpression is an unfavorable prognostic biomarker for NSCLC patients3.1 Overall survival and prognostic factors for total patientsThe deadline for follow-up study was December 31,2014. The follow-up period was range from 5 months to 60 months, with a median follow-up period of 33 months. For all of the included patients, median overall survival were 37.0 months (95%CI:32.3-41.7 months), the one-year survival rate is 91.4%, three-year survival rate is 53.2%, and five-year survival rate is 40.3%. The median survival of these patients with higher expression of CCNB2 was 17.0 months (95%CI: 16.3-17.7months),while for that of lower, the survival was 40.0 months (95%CI: 34.9-45.1 months), which is statistically significant (P=0.000).Univariate analysis revealed that the factors that influenced the overall survival of NSCLC patients were CCNB2 (p=0.000),TNM stage (p=0.000), tumor size (T stage) (p=0.000), Lymph node metastasis (N stage) (p=0.000) and distant metastasis (P=0.000), while factors, such as sex, age, smoking status, pathological type, degree of differentiation had no impact on survival.Multivariate COX analysis indicated that only CCNB2 expression (HR=2.27,95%CI:1.40-3.69,P=0.001), T stage (HR=1.87,95%CI: 1.23-2.84,P=0.003), N stage (HR=2.16,95%CI:1.10-4.23,p=0.025) and M stage (HR=2.95,95%CI:1.45-6.01,/>=0.003) were independent prognostic factors. Of particular note was TNM stage was not an independent predictor which was contrary to our knowledge, and its cause may be kinds of bias in the collection of data.3.2.Stratification analysis of overall survival for CCNB2We stratified by sex, age, smoking, pathological type, degree of differentiation, TNM stage, tumor size, lymph node metastasis, and distant metastasis to compare the survival of patients between higher expression group and lower expression group, and found that, except for the elder, high degree of differentiation, and early N stage subgroup, for all of the other subgroups, the survival of patients with higher expression of CCNB2 is shorter than that of lower.3.3. Overall survival and prognostic factors for patients with MO stageThe deadline for follow-up study was December 31,2014. The follow-up period was range from 5 months to 60 months, with a median follow-up period of 34 months. For the 171 patients with MO stage, median overall survival was 40.0 months (95%CI:34.9-45.1 months). The median survival of these patients with higher expression of CCNB2 was 31.0 months (95%CI:27.8-34.2 months),while for that of lower, the survival was 48.9 months (95%CI:45.5-52.2 months), which is statistically significant (P=0.000).Univariate analysis revealed that the factors that influenced the overall survival of NSCLC patients were CCNB2 (p=0.000),TNM stage (p=0.000), tumor size (T stage) (p=0.000) and Lymph node metastasis (N stage) (p=0.000), while factors, such as sex, age, smoking status, pathological type, degree of differentiation had no impact on survival.Multivariate COX analysis indicated that only CCNB2 expression (HR=2.25,95%CI:1.38-3.66,P=0.001), T stage (HR=1.87,95%CI:1.23-2.84, p=0.003) and N stage (HR=2.16,95%CI:1.10-4.23, P=0.025) were independent prognostic factors. It was worth mentioning TNM stage was not an independent predictor which was contrary to popular belief, and we speculated that it may be ascribed to kinds of bias in data collection.Conclusion1. CCNB2 probablely promotes the genesis and development of non-small cell lung cancer;2.Compared with the normal tissues adjacent to cancer, the expression of CCNB2 in lung cancer tissue is higher;3. CCNB2 expression is closely correlated with degree of differentiation, tumor size, Lymph node metastasis, distant metastasis and TNM staging;4. For total 186patients,the survival of patients with higher expression of CCNB2 is shorter than that of lower expression of CCNB2; Furthermore, tumor size, Lymph node metastasis, distant metastasis and TNM staging are prognostic factors in NSCLC patients; however, none of sex, age, smoking status, pathological type and degree of differentiation is prognostic factors.5.Westratifiedby sex, age, smoking, pathological type, degree of differentiation,TNM stage, tumor size, lymph node metastasis, and distant metastasis to compare the survival of patients between higher expression group and lower expression group, and found that, except for the elder, high degree of differentiation, and early N stage subgroup, for all of the other subgroups, the survival of patients with higher expression of CCNB2 is shorter than that of lower.6. For total 171 patients of stage M0,the survival of patients with higher expression of CCNB2 is also shorter than that of lower expression of CCNB2; Moreover, tumor size and Lymph node metastasisare prognostic factors in NSCLC patients; however, none of sex, age, smoking status, pathological type and degree of differentiation is prognostic factors.