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，即新生毛细血管的形成，是肿瘤进展的必要条件。我们曾报道过1型血管紧张素受体（AT1-R）拮抗剂可减少肿瘤相关血管生成。由于有报道称抗血管生成药物可以提高放射治疗的疗效，我们在这里测试了AT1-R阻断是否会促进放射治疗的效果。方法：将1 × 10^6个LLC细胞注射到雄性C57BL/6小鼠皮下，当肿瘤平均体积达到0.1 cm3左右时，第1天给予3、5、10、15 Gy的辐射剂量。结果：第22天平均肿瘤体积分别为6.39 （3 Gy）、6.15 （5 Gy）、5.15 （10 Gy）、3.07 (15 Gy) cm3。10 Gy联合AT1R拮抗剂TCV-116 （30 mg/kg）对肿瘤生长的抑制作用较对照（8.81 + 0.45 cm^3）显著提高83% （1.47 + 0.11 cm^3, P < 0.01）。平均血管密度也是如此，并且联合…More治疗显著降低了肿瘤相关血管生成。CD31的表达减少证实了这一点。抗血管内皮生长因子（VEGF）中和抗体阻断LLC肿瘤生长。实时荧光定量PCR分析显示，与对照组小鼠相比，联合治疗小鼠的VEGF明显减少。结论：这些结果表明，放射联合AT1-R阻断显著降低了LLC生长速率，这是由于通过降低VEGF水平来减少新生血管。放射与AT1R阻断联合治疗可能成为一种有效的肿瘤治疗新策略。2)我们在此分析了6例吉非替尼反应的肺癌治疗前和治疗后匹配肿瘤中的EGFR突变。通过常规pcr测序，在每个病例的预处理样本中检测到经典突变，并且在4例病例的治疗病变中很容易确认相同的突变，而在病例1和病例2中没有相应的突变。随后的突变体富集肽核酸介导、PCR夹夹和亚克隆试验在病例1的治疗病变的小细胞中检测到突变，但在病例2中仍然未能检测到突变。因此，我们对预处理进行了基于微解剖的细胞簇突变分析，发现包括前两个在内的三个同时含有突变型或野生型egfr的肿瘤细胞，尽管后者仅占一小部分。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；吉非替尼敏感突变体减少或消失，而野生克隆选择性地在吉非替尼治疗下存活。此外，在3例治疗的病灶中，有一小部分检测到继发性T790M。因此，EGFR遗传异质性背景下的选择也可能有助于在一定比例的非小细胞肺癌中获得吉非替尼耐药性。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