Clinical and molecular histo-pathological research on predictive evaluation of the effect of radiotherapy combined with or without molecular targeting agents for lung cancer

1)目的：血管生成，即新的毛细血管的形成，对肿瘤的发展至关重要。我们曾报道，1型血管紧张素受体(AT1-R)拮抗剂减少了肿瘤相关血管的生成。由于抗血管生成药物被报道可以提高放射治疗的疗效，我们在这里测试了AT1-R阻滞剂是否有助于放射治疗的效果。方法：1将1×10~(-6)LLC细胞接种于雄性C57BL/6小鼠皮下，当肿瘤平均体积达到0.1cm3左右时，第1天给予3，5，10，15Gy4个剂量的放射治疗。结果：第22天平均肿瘤体积分别为6.39(3，5)，6.15(5)，5.15(10)，3.07(15)cm3。10Gy射线联合AT1R拮抗剂TCV-116(30 mg/kg)对肿瘤生长的抑制率为83%(1.47±0.11 cm^3，P&lt；0.01)，与对照组(8.81±0.45 cm^3)相比，差异有统计学意义(P<0.01)。平均血管密度和TH…的组合也是如此更有效的治疗可显著减少肿瘤相关的血管生成。CD31的表达减少证实了这一点。用抗血管内皮生长因子(VEGF)中和抗体阻断LLC肿瘤生长。实时聚合酶链式反应分析显示，与对照组相比，联合治疗组小鼠的血管内皮生长因子水平显著降低。结论：辐射联合AT1-R阻断剂可显著降低LLC的生长速度，其机制可能是通过降低VEGF水平来减少新生血管的生成。放射和AT1R阻断的联合治疗可能成为一种有效的癌症治疗新策略。2)我们分析了6例吉非替尼敏感的肺癌患者治疗前后相匹配的肿瘤中的EGFR突变。用常规的基于聚合酶链式反应的测序，在每个病例的预处理样本中都检测到典型的突变，而在病例1和病例2的治疗皮损中可以很容易地证实相同的突变，而相应的突变在病例1和病例2中是不存在的。随后的突变富集肽-核酸介导、PCR钳制和亚克隆试验在病例1的治疗病变的小细胞中检测到了突变，但在病例2中仍然没有检测到突变。因此，我们进行了基于显微切割的预处理细胞团突变分析，发现3个，包括前两个同时含有突变的或野生的-EGFR的肿瘤细胞，尽管后者只占很小的一部分。这些发现表明，一些非小细胞肺癌在EGFR突变方面具有遗传异质性；对吉非替尼敏感的突变体减少或消失，而野生克隆在吉非替尼治疗下选择性存活。此外，在3例患者的一小部分治疗皮损中检测到继发性T790M。因此，在一定比例的非小细胞肺癌中，基于EGFR基因异质性的选择也可能有助于获得吉非替尼耐药性。较少

1) Objective: Angiogenesis, the formation of new capillary blood vessels, is essential for tumor progression. We had reported that Type 1 angiotensin receptor (AT1-R) antagonist reduced tumor-associated angiogenesis. Since anti-angio-genic agents were reported to enhance efficacy of radiation therapy, we tested here whether or not AT1-R blockade facilitates the effects of radiation. Methods: 1 X 10^6 LLC cells were injected into the subcutaneous tissue of male C57BL/6 mice, and when the average tumor volume reached around 0.1 cm3, radiation doses (3, 5, 10, and 15 Gy) were given on day 1. Results: The mean tumor volumes at day 22 were 6.39 (3 Gy), 6.15 (5 Gy), 5.15 (10 Gy), and 3.07 (15 Gy) cm3, respectively. Combination of 10 Gy radiation with AT1R antagonist TCV-116 (30 mg/kg) significantly inhibited tumor growth by 83% (1.47 + 0.11 cm^3, P < 0.01) in comparison with its inhibition of control tumors (8.81 + 0.45 cm^3). The same was true for mean vessel density, and the combination th … More erapy markedly reduced tumor-associated angio- genesis. This was confirmed by the reduced expression of CD31. LLC tumor growth was blocked by neutralizing antibody against vascular endothelial growth factor (VEGF). Real-time PCR analysis of VEGF disclosed a marked reduction in the mice under combination therapy, compared with control mice. Conclusions: These results suggest that combination of radiation with AT1-R blockade markedly reduced the LLC growth rate, and that this was due to reduction of neovascularization by reducing VEGF levels. Combination therapy consisting of radiation and AT1R blockade may become an effective novel strategy for cancer treatment.2) We here analyzed EGFR mutations in matched pre- and post-therapeutic tumors of six gefitinib-responding lung cancers. With conventional PCR-based sequencing, classic mutations were detected in pretreatment samples of each case, and the same mutations could be readily confirmed in treated lesions of four cases, While the corresponding mutations were absent in those of cases 1 and 2. Subsequent mutant-enriched peptide-nucleic-acid-mediated, PCR clamping and subcloning assays detected the mutation in minor cells of treated lesions of case 1, but still failed top detect mutation in case 2. We thus performed a microdissection-based cell cluster mutation analysis of pretreatment and found that three, including the first two concurrently contained tumor cells with either mutant- or wild-EGFR, although the latter composed only a minor fraction. These findings suggest that some NSCLC are genetically heterogeneous with regard to EGFR mutations; the gefitinib-sensitive mutants decrease or vanish while the wild clones selectively survive with gefitinib treatment. In addition, secondary T790M was detected in a small fraction of treated lesions of three cases. Thus, selection on a background of EGFR genetic heterogeneity may also contribute to acquisition of gefitinib resistance in a proportion of non-small-cell lung carcinomas. Less