Biomarkers of Human Lung Cancer

人类肺癌的生物标志物

• 批准号: 8349212
• 负责人: Curtis Harris
• 金额: $ 60.13万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349212
• 关键词: 19p; 19p13.3; Accounting; Adenocarcinoma; Adjuvant; Adjuvant Chemotherapy; Alleles; Anabolism; Biological Markers; Cancer cell line; Cancerous; Cells; Chromosomes; Clinical; Clinical Trials; Code; Coupled; DNA; DNA copy number; Data; Development; Diagnosis; Diagnostic; Diagnostic Neoplasm Staging; Disease; Early Diagnosis; Estrogen Receptor alpha; Estrogen Receptors; Estrogens; Family; Female; Formalin; Freezing; Gender; Genes; Genetic; Genetic Polymorphism; Genetic Transcription; Germ Lines; Goals; Histocompatibility Testing; Hormone Receptor; Hormone replacement therapy; Human; In Vitro; International; Japan; Japanese Population; Lead; Loss of Heterozygosity; Lung Adenocarcinoma; Lung Neoplasms; Malignant Neoplasms; Malignant Pleural Mesothelioma; Malignant neoplasm of lung; Maps; Maryland; Measures; Messenger RNA; MicroRNAs; Molecular; Mutation; Non-Small-Cell Lung Carcinoma; Normal tissue morphology; Norway; Oncogenic; Outcome; Paraffin Embedding; Pathway interactions; Patients; Plasma; Pleural Mesothelial Cell; Pleural Mesothelioma; Predictive Value; Progesterone; Protein-Serine-Threonine Kinases; RNA; Randomized; Relapse; Reporting; Resected; Role; STK11 gene; Sampling; Serum; Simian virus 40; Single Nucleotide Polymorphism; Smoker; Somatic Mutation; Specimen; Staging; Systems Biology; TNM; Telomerase; Telomerase Inhibitor; Telomere Length Maintenance; Testing; Tissues; Translations; Tumor Suppressor Genes; Tumor stage; Variant; base; burden of illness; cancer initiation; cancer therapy; carcinogenesis; chromosome loss; cohort; improved; in vitro Model; in vivo; male; mortality; novel; outcome forecast; prognostic; telomere; therapeutic target; tumor; tumor progression; tumorigenesis

Early detection of primary lung cancer is difficult yet important since diagnosis at earlier stages is associated with significantly better survival. Circulating microRNA (miR) profiles have been proposed as promising diagnostic and prognostic biomarkers for cancer, including lung cancer. The goal in this study was to establish biomarkers for early detection of primary lung cancer (NSCLC) by measuring circulating miRs. We looked at paired serum and plasma samples of patients with early stage NSCLC and matched controls. We discovered differential expression of the following miRs: miR-146b, miR-221, let-7a, miR-155, miR-17-5p, miR-27a, and miR-106a, which were significantly reduced in the serum of NSCLC cases, while miR-29c was significantly increased. Even though we did not observe any significant differences in plasma, and expression levels in serum did not correlate well with levels of plasma, these tissue types are obtained differently, which could account for differences in the results. Moreover, reduced plasma expression of let-7b was modestly associated with worse cancer-specific mortality in all patients, whereas reduced serum expression of miR-223 was modestly associated with cancer-specific mortality in stage IA/B patients. Additionally, we looked at the expression of biologically relevant miRs (miR-21, miR-29b, miR-34a/b/c, miR-155, and let-7a) in formalin-fixed paraffin-embedded tumor specimens from the patients who participated in the International Adjuvant Lung Cancer Trial (IALT), the largest randomized study conducted to date of adjuvant chemotherapy in patients with radically resected NSCLC. The goal was to evaluate if the expression of these miRs represents prognostic and predictive value for survival. Importantly, the expression of certain miRs has been associated with chemoresistance. However, our results indicate that the miRs expression patters examined were neither predictive nor prognostic in this large patient cohort. Based on the previous miR microarray data of lung cancer specimens from NSCLC adenocarcinoma patients, we investigated the expression of 5 miRs whose expression were significantly altered in lung cancer and were associated with cancer-specific mortality. Three of those miRs (miR-17, miR-21, and miR-155) were potential oncogenic miRs, showing increased expression in tumors with high expression levels being associated with poor prognosis. We investigated their expression in early-stage lung adenocarcinoma snap-frozen tissues from NSCLC patients that originated from Maryland, Norway and Japan in association with tumor progression and survival. We found elevated expression of all three miRs to be associated with worse cancer-specific mortality in the Maryland cohort. However, only miR-21 showed the same association in the Norwegian cohort and worse relapse-free survival in the Japanese cohort. Additionally, more advanced stage tumors expressed significantly higher levels of miR-21 compared with TNM stage I patients, in which group miR-21 was also associated with worse cancer-specific mortality and relapse-free survival, independent of other clinical factors. We found that elevated miR-21 expression is independently associated with worse prognosis in 3 independent NSCLC adenocarcinoma cohorts from different regions of the world. This finding has a translational relevance; miR-21 expression has potential utility as an early-stage prognostic biomarker for NSCLC adenocarcinoma patients. Although we did not observe a significant increase of miR-21 expression in FFPE tissues, this could be due to lower quality of RNA extracted from those tissues, as has been reported previously. Moreover, we have undertaken a systems biology approach to study the effect of miRs in cancer. Each miR can control translation of hundreds of different coding messengers, and a single messenger can be controlled by more than one miR. We combined differential expression, genetic networks and DNA copy number alterations. We confirmed, or discovered, miRs with comprehensive roles in cancer: has-miR-103/106 were downregulated in cancer, whereas has-miR-30 became most prominent; has-miR-17/92 family was amplified and the has-miR-143/145 cluster deleted in cancer; has-miR-30 and has-miR-204 were found to be physically altered at the DNA copy number level. We reported the first miR network from normal tissues, but also built miR networks for coupled cancerous and noncancerous tissues, and identified cancer variations in miR networks. Finally, we superimposed DNA variations onto expression data to generate a comprehensive miR alteration map in cancer. In addition to our studies with miRs as important biomarkers in lung cancer, we investigated a tumor suppressor gene, LKB1/STK11, which has a serine-threonine kinase activity and is located on chromosome 19p13.3. Chromosome 19p is the second most common region of loss in lung tumors of smokers, and LKB1 mutations are found in 30% of lung cancer cell lines and in a smaller portion of primary lung adenocarcinomas. In this study, we used several complementary genetic approaches to assess the KLB1 locus in primary NSCLC. Our data identified total inactivation of the LKB1 gene by either homozygous deletion (HD) or loss of heterozygosity (LOH) with somatic mutation in 39% of tested samples, whereas loss of chromosome 19p region by HD or LOH at the LKB1 region occurred in 90% of NSCLC. Furthermore, we investigated the role of tumor estrogen receptors (ERs) and serum estrogen in lung cancer with a hypothesis that ERs and functional single nucleotide polymorphisms (SNPs) in the estrogen biosynthesis pathway are associated with poorer lung cancer survival. Serum estrogen, progesterone, tumor messenger RNA expression of hormone receptors and germ line DNA polymorphisms were analyzed for associations with lung cancer survival. Patients in the highest tertile of serum estrogen had worse survival in all three cohorts investigated in this study. Furthermore, the variant allele of estrogen receptor alpha (ER-alpha) polymorphism (rs2228480) was significantly associated with increased tumor ER-alpha levels and worse survival in all cohorts. Results were independent of gender and hormone replacement therapy. Thus, we reported a significant association of increased serum estrogen with poorer survival among lung cancer male and female patients. Additionally, we investigated a very important mechanism, cellular immortalization, which is one of the hallmarks of cancer and is dependent on the activity of a telomere length maintenance mechanism (TMM), either telomerase or alternative lengthening of telomeres (ALT). We examined 43 malignant pleural mesotheliomas (MPMs). The TMMs are widely regarded as potential targets for cancer therapies and telomerase inhibitors have entered clinical trials. We asked what proportion of MPMs use ALT and/or telomerase. We found that 43 of 43 MPMs were telomerase-positive[+] and Alt-negative[-]. To investigate whether MPM cells are unusually susceptible to activation of telomerase, we examined activation of the TMMs in an in vitro model of cellular immortalization, in which normal pleural mesothelial cells were transduced with SV40. We discovered that pleural mesothelioma cells are capable of activating either TMM in vitro, and the observation that 100% MPMs were telomerase[+] suggests that there are factors in vivo that select for telomerase activity during oncogenesis of this tumor type. We concluded that MPM is a tumor that could be considered for anti-telomerase therapy. Genes and mechanisms described in our studies, important to lung cancer initiation, progression and outcome, present potential molecular prognostic and diagnostic biomarkers in lung cancer.

原发性肺癌的早期检测虽然困难但也很重要，因为早期诊断与显着提高的生存率相关。循环 microRNA (miR) 谱已被提议作为癌症（包括肺癌）的有前途的诊断和预后生物标志物。本研究的目标是通过测量循环 miR 建立用于早期检测原发性肺癌 (NSCLC) 的生物标志物。我们研究了早期 NSCLC 患者和匹配对照的配对血清和血浆样本。我们发现以下miR的差异表达：miR-146b、miR-221、let-7a、miR-155、miR-17-5p、miR-27a和miR-106a在NSCLC病例的血清中显着降低，而miR-29c显着增加。尽管我们没有观察到血浆中的任何显着差异，并且血清中的表达水平与血浆水平没有很好的相关性，但这些组织类型的获得方式不同，这可以解释结果的差异。此外，let-7b 的血浆表达降低与所有患者的癌症特异性死亡率较差有一定的相关性，而 miR-223 的血清表达降低与 IA/B 期患者的癌症特异性死亡率有一定的相关性。此外，我们还观察了参与国际肺癌辅助试验 (IALT) 的患者福尔马林固定石蜡包埋的肿瘤标本中生物学相关 miR（miR-21、miR-29b、miR-34a/b/c、miR-155 和 let-7a）的表达，该试验是迄今为止对根治性切除的 NSCLC 患者进行辅助化疗的最大随机研究。目的是评估这些 miR 的表达是否代表生存的预后和预测价值。重要的是，某些 miR 的表达与化疗耐药相关。然而，我们的结果表明，在这个大型患者队列中，检查的 miR 表达模式既不能预测也不能预后。基于之前 NSCLC 腺癌患者肺癌标本的 miR 微阵列数据，我们研究了 5 个 miR 的表达，这些 miR 的表达在肺癌中显着改​​变，并且与癌症特异性死亡率相关。其中三种 miR（miR-17、miR-21 和 miR-155）是潜在的致癌 miR，显示肿瘤中表达增加，高表达水平与不良预后相关。我们研究了它们在来自马里兰州、挪威和日本的非小细胞肺癌患者的早期肺腺癌速冻组织中的表达与肿瘤进展和生存的关系。我们发现，在马里兰队列中，所有三种 miR 的表达升高都与较差的癌症特异性死亡率相关。然而，只有 miR-21 在挪威队列中显示出相同的关联，而在日本队列中则显示出较差的无复发生存率。此外，与 TNM I 期患者相比，晚期肿瘤的 miR-21 表达水平显着更高，其中 miR-21 还与较差的癌症特异性死亡率和无复发生存率相关，且与其他临床因素无关。我们发现，在来自世界不同地区的 3 个独立 NSCLC 腺癌队列中，miR-21 表达升高与较差的预后独立相关。这一发现具有转化意义； miR-21 表达具有作为 NSCLC 腺癌患者早期预后生物标志物的潜在用途。尽管我们没有观察到 FFPE 组织中 miR-21 表达显着增加，但这可能是由于从这些组织中提取的 RNA 质量较低，正如之前报道的那样。此外，我们采用系统生物学方法来研究 miR 在癌症中的作用。每个miR可以控制数百个不同编码信使的翻译，并且单个信使可以由多个miR控制。我们将差异表达、遗传网络和 DNA 拷贝数改变结合起来。我们证实或发现了 miR 在癌症中具有全面的作用：has-miR-103/106 在癌症中下调，而 has-miR-30 最为突出； has-miR-17/92 家族在癌症中被扩增，并且 has-miR-143/145 簇被删除；发现 has-miR-30 和 has-miR-204 在 DNA 拷贝数水平上发生了物理改变。我们报告了第一个来自正常组织的 miR 网络，但也为耦合的癌性和非癌性组织构建了 miR 网络，并鉴定了 miR 网络中的癌症变异。最后，我们将 DNA 变异叠加到表达数据上，生成癌症中全面的 miR 改变图。除了将 miR 作为肺癌重要生物标志物的研究之外，我们还研究了肿瘤抑制基因 LKB1/STK11，该基因具有丝氨酸-苏氨酸激酶活性，位于染色体 19p13.3 上。 19p 染色体是吸烟者肺肿瘤中第二常见的缺失区域，LKB1 突变存在于 30% 的肺癌细胞系和一小部分原发性肺腺癌中。在这项研究中，我们使用了几种互补的遗传学方法来评估原发性 NSCLC 中的 KLB1 基因座。我们的数据发现，在 39% 的测试样本中，LKB1 基因因纯合性缺失 (HD) 或杂合性缺失 (LOH) 以及体细胞突变而完全失活，而 90% 的 NSCLC 中则发生了 HD 或 LKB1 区域 LOH 造成的染色体 19p 区域缺失。此外，我们研究了肿瘤雌激素受体（ER）和血清雌激素在肺癌中的作用，假设雌激素生物合成途径中的ER和功能性单核苷酸多态性（SNP）与较差的肺癌生存相关。分析血清雌激素、孕激素、激素受体的肿瘤信使 RNA 表达和种系 DNA 多态性与肺癌生存的关系。在本研究调查的所有三个队列中，血清雌激素水平最高的患者的生存率较差。此外，雌激素受体α（ER-α）多态性（rs2228480）的变异等位基因与所有队列中肿瘤ER-α水平升高和生存率较差显着相关。结果与性别和激素替代疗法无关。因此，我们报告了血清雌激素增加与肺癌男性和女性患者较差的生存率存在显着相关性。此外，我们研究了一种非常重要的机制，即细胞永生化，它是癌症的标志之一，并且依赖于端粒长度维持机制（TMM）的活性，即端粒酶或端粒选择性延长（ALT）。我们检查了 43 例恶性胸膜间皮瘤 (MPM)。 TMM被广泛认为是癌症治疗的潜在靶点，端粒酶抑制剂已进入临床试验。我们询问 MPM 使用 ALT 和/或端粒酶的比例是多少。我们发现 43 个 MPM 中有 43 个呈端粒酶阳性 [+] 和 Alt 阴性 [-]。为了研究 MPM 细胞是否对端粒酶的激活异常敏感，我们在细胞永生化的体外模型中检查了 TMM 的激活，其中用 SV40 转导正常胸膜间皮细胞。我们发现胸膜间皮瘤细胞能够在体外激活任一 TMM，并且观察到 100% MPM 是端粒酶[+]，这表明体内存在一些因素在这种肿瘤类型的肿瘤发生过程中选择端粒酶活性。我们得出的结论是，MPM 是一种可以考虑进行抗端粒酶治疗的肿瘤。我们的研究中描述的基因和机制对肺癌的发生、进展和结果很重要，为肺癌提供了潜在的分子预后和诊断生物标志物。