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，即新毛细血管的形成，是肿瘤进展所必需的。我们曾报道1型血管紧张素受体（AT 1-R）拮抗剂可减少肿瘤相关的血管生成。由于抗血管生成剂的报道，以提高放射治疗的疗效，我们在这里测试是否AT 1-R阻断促进辐射的影响。研究方法：将1 X 10^6个LLC细胞注射到雄性C57 BL/6小鼠的皮下组织中，当平均肿瘤体积达到约0.1 cm 3时，在第1天给予辐射剂量（3、5、10和15戈伊）。结果：第22天的平均肿瘤体积分别为6.39（3戈伊）、6.15（5戈伊）、5.15（10戈伊）和3.07（15戈伊）cm 3。10戈伊照射联合AT 1受体拮抗剂TCV-116（30 mg/kg）对肿瘤生长的抑制率为83%（1.47 ± 0.11 cm ^3，P < 0.01），而对照组为8.81 ± 0.45 cm ^3。平均血管密度也是如此， 关于我们 雌二醇显著减少肿瘤相关的血管生成。这通过CD 31的表达减少得到证实。LLC肿瘤生长被血管内皮生长因子（VEGF）的中和抗体阻断。与对照组小鼠相比，VEGF的实时PCR分析显示联合治疗组小鼠的VEGF显著降低。结论：这些结果表明，辐射与AT 1-R阻断的组合显著降低了LLC的生长速率，这是由于通过降低VEGF水平减少了新血管形成。联合放疗和AT 1 R阻断治疗可能成为一种有效的癌症治疗新策略。2）我们在此分析了6例吉非替尼反应肺癌治疗前后的EGFR突变。采用常规PCR测序方法，在每个病例的治疗前样本中检测到经典突变，并且在4个病例的治疗病灶中可以容易地确认相同的突变，而在病例1和病例2中不存在相应的突变。随后的突变富集肽核酸介导的PCR钳位和亚克隆测定检测到病例1的治疗病变的小细胞中的突变，但仍未能检测到病例2的最高突变。因此，我们进行了基于显微解剖的细胞簇突变分析的预处理，发现三个，包括前两个同时含有突变或野生型EGFR的肿瘤细胞，尽管后者只占一小部分。这些结果表明，一些NSCLC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，而野生型克隆选择性地在吉非替尼治疗后存活。此外，继发性T790 M检测到一小部分治疗病变的3例。因此，在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。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