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

为了预测放射治疗后局部控制率主要相关的辐射敏感性，研究了辐射反应与癌基因、肿瘤细胞凋亡相关基因表达和低氧相关蛋白等生物学参数的相关性。对人体标本进行免疫组织化学研究和各种DNA/RNA分析，以获得与辐射敏感性相关的参数，并以肿瘤细胞系和实验动物为材料进行分析。以c-erb B-2癌蛋白表达、p21、p27、p53蛋白表达、血小板源性内皮细胞生长因子、诱导型一氧化氮合酶和缺氧诱导因子-1α(HIF-1α)为指标分析预后。71例患者中，PD-ECGF阴性31例，阳性40例。…的5年存活率PD-ECGF阳性患者多于PD-ECGF阴性患者(p=0.026)。INOS阳性的癌细胞呈胞浆染色。INOS阴性41例，阳性27例。INOS阴性患者与iNOS阳性患者的预后无明显相关性。c-erbB-2/HER2阳性表达见于癌细胞膜和胞浆。C-erb-B2/HER2阳性9例，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%，较阴性患者的52%预后更差(p=0.10)。COX比例风险模型多因素分析显示，c-erb B-2/hER2是独立的预后因素(p=0.024)，p21、p27和p53阳性的癌细胞和放疗所致的癌变或肿胀的癌细胞具有核内反应性。放疗前平均P27标记指数在27Gy时显著降低，而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瘤无转移生存期显著相关。放疗前P53标记指数高的患者总体存活率较低。放疗前2例标本均未检出p53基因突变(外显子5-8)，提示为野生型。在38例患者中，HIF-1α高表达17例(45%)，P53高表达22例(58%)，bax高表达15例(39%)，bcl-2高表达15例(39%)，28例(74%)阳性。HIF-1α高表达与肿瘤复发呈显著正相关(p&lt；0.05)。此外，低氧诱导因子-1α与无复发生存率显著相关(p=0.0 4)。HIF-1α高表达与局部控制率差异无统计学意义(P=0.17)，而HIF-1α高表达预测远处转移有显著意义(P=0.03)。相反，其他因素对IIIB期宫颈癌患者的临床预后没有影响。结论：癌细胞中PD-ECGF阳性被认为是单纯放射治疗的宫颈鳞癌预后良好的预测因素。C-erb B-2/HER2在宫颈肿瘤中的表达可作为预测晚期宫颈癌放疗后PALNM预后的指标。放疗前癌细胞中p27的高表达和p53的低表达是预测单纯放疗宫颈鳞癌患者预后的良好因素。P27和P53指数在放疗前至27Gy时呈倒转变化，提示放射治疗作用之一。P53癌基因在27Gy时将放疗前野生型P53转变为突变型P53的现象可能是辐射效应。低氧诱导因子-1α是预测宫颈鳞癌放疗后转移的重要预后因子。较少

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