Basic and Clinical Investigation for Modulation of Radiation Sensitivity in Radiation Therapy combined by Genetherapy

研究了放射反应与肿瘤癌基因、肿瘤细胞凋亡相关基因表达和缺氧相关蛋白等多种生物学参数的相关性，以预测放射治疗后放射敏感性主要与局部控制概率有关。对人体标本进行免疫组化研究和各种DNA/RNA分析，获得与辐射敏感性相关的参数，并对肿瘤细胞系和实验动物进行分析。通过c-erbB-2癌蛋白表达（CerbB-OE）、p21、p27、p53蛋白表达、血小板源性内皮细胞生长因子（PD-ECGF）、诱导NOS （iNOS）、缺氧诱导因子-1α (HIF-1α)等指标分析预后。PD-ECGF阳性的癌细胞显示核内和细胞质染色模式。71例患者中，PD-ECGF阴性31例，阳性40例。pd - ecgf阳性患者的5年生存率明显高于pd - ecgf阴性患者（p=0.026）。iNOS阳性的癌细胞呈细胞质染色模式。iNOS阴性41例，阳性27例。inos阴性与inos阳性患者预后无显著相关性，癌膜和细胞质中可见c- erbb -2/HER2阳性染色。9例标本c- erbb - b2 /HER2阳性，总阳性率为43%。所有患者的5年生存率为38%。c-erbB-2/HER2阳性患者的5年生存率为28%，比阴性患者的52%有更差的预后趋势（p=0.10）。Cox比例风险模型多因素分析显示，c-erbB-2/HER2是独立预后因素（p=0.024）。p21、p27和p53呈阳性的癌细胞和放疗引起的变性或肿胀的癌细胞表现出核内反应性。放疗前平均p27标记指数在27Gy时显著降低，反之，平均p53标记指数在27Gy时显著升高。放疗前高p27标记指数与良好的无病和无转移生存率显著相关。放疗前p53标记指数高，总生存期差。放疗前2例患者均未见p53（5-8外显子）突变，提示为野生型。在27Gy下，5个样本中有4个显示p53突变（外显子5和7），其余1个没有突变。38例患者中HIF-1α、p53、bax和bcl-2分别高表达17例（45%）、22例（58%）、15例（39%）和15例（39%），HPV感染阳性28例（74%）。HIF-1α高表达与疾病复发有显著正相关（p<0.05）。HIF-1α与无复发生存率有显著相关性（p=0.04）。HIF-1α高表达与局部控制率之间无统计学意义（p=0.17），而HIF-1α水平预测远处转移有很强的统计学意义（p=0.03）。相反，其他因素对IIIB期宫颈癌患者的临床结局没有影响。肿瘤细胞pd - ecgf阳性被认为是宫颈鳞癌单独放疗预后良好的预测因素。宫颈肿瘤c-erbB-2/HER2表达可能是晚期宫颈癌PALNM放疗后预后的预测指标。放疗前癌细胞中p27的高表达和p53的低表达是单独放疗宫颈鳞状细胞癌患者预后的预测良好因素。p27和p53指数从放疗前到27Gy时的周期变化呈逆转模式，提示放疗有一定效果。在27Gy照射下，p53癌基因由放疗前的野生型变为突变型的现象可能是辐射效应所致。HIF-1α是一个重要的预后因素，特别是用于预测IIIB期宫颈鳞状细胞癌放射治疗后的未来转移。少

Correlations between radiation response and various biological parameters including oncogenes, apoptosis relating gene expressions of tumors and hypoxia relating proteins were investigated for prediction of radiation sensitivity predominantly relating local control probability following radiation therapy.Immunohistochemical study and various DNA/RNA analysis for human specimens were performed for obtaining parameters relating to radiation sensitivity as well as analysis for tumor cell lines and experimental animals as materials. Prognosis was analyzed by various parameters including c-erbB-2 oncoprotein expression (CerbB-OE), p21, p27, p53 protein expression, Platelet-derived endothelial cell growth factor (PD-ECGF), inducible NOS (iNOS) and Hypoxia-inducible factor-1α (HIF-1α).RESULTS. Cancer cells that were positive for PD-ECGF showed intranuclear and cytoplasmic staining patterns. Of the 71 patients, 31 were negative for PD-ECGF and 40 were positive. The 5-year survival rate of the … More PD-ECGF-positive patients was significantly better than that of the PD-ECGF-negative patients (p=0.026). Cancer cells that were positive for iNOS showed a cytoplasmic staining pattern. Forty one patients were negative for iNOS and 27 were positive. No significant prognostic correlation existed between iNOS-negative and iNOS-positive patients.c-erbB-2/HER2 positive staining was observed in cancer membrane and cytoplasm. Nine specimens were positive for c-erb-B2/HER2 and the total positive rate was 43%. The 5-year survival rate was 38% for all patients. The 5-year survival rate of the c-erbB-2/HER2 positive patients was 28%, representing a trend toward poorer prognosis than the 52% of negative patients (p=0.10). Multivariate analysis using Cox proportional hazards model showed that c-erbB-2/HER2 was an independent prognostic factor (p=0.024).Cancer cells, and degenerated or swollen cancer cells caused by radiotherapy that were positive for p21, p27, and p53 showed an intranuclear reactivity. The mean p27-labeling index before radiotherapy was significantly decreased at 27Gy, conversely, the mean p53-labeling index was significantly increased at 27Gy. The high p27-labeling index before radiotherapy was significantly associated with good disease free and metastasis free survivals. The high p53-labeling index before radiotherapy was a poor overall survival. All the 2 samples before radiotherapy showed no mutations of p53 (exons 5-8), that implied the wild type. Four out of 5 samples at 27Gy showed mutations of p53 (exons 5 and 7), and the remaining one had no mutations.Of 38 patients, high expression of HIF-1α, p53, bax, and bcl-2 were seen in 17 (45%), 22 (58%), 15 (39%), and 15 (39%) patients, respectively, and 28 patients (74%) showed the positive infection with HPV. There was a significant positive correlation between high HIF-1α expression and disease recurrence (p<0.05). Furthermore, HIF-1α had a significant correlation with the recurrence free survival rate (p=0.04). No statistical significance was noted between high HIF-1α expression and local control rate (p=0.17), whereas HIF-1α status predicted distant metastasis with strong significance (p=0.03). Conversely, other factors demonstrated no impact on clinical outcome of patients with stage IIIB cervical carcinoma.CONCLUSIONS. PD-ECGF-positivity in cancer cells is regarded as a predictive factor for good prognosis in cervical squamous cell carcinoma treated with radiotherapy alone. c-erbB-2/HER2 expression of cervical tumors might be a predictive parameter of the prognosis after radiation therapy for PALNM of advanced uterine cervical carcinoma patients. The high p27 and low p53 expression in cancer cells before radiotherapy is a predictive good factor for the prognosis of patients with cervical squamous cell carcinoma treated with radiotherapy alone. The p27 and p53 indexes showed reversal patterns of changes in periods from before radiotherapy to at 27Gy, implying one of the radiotherapeutic effects. The phenomenon of the p53 oncogene changing the wild type of p53 before radiotherapy into the mutant type at 27Gy may be the radiation effect. HIF-1α is an important prognostic factor, especially for predicting future metastasis after radiation therapy for patients with Stage IIIB squamous cell carcinoma of the cervix. Less