Biomarkers of Human Lung Cancer

早期发现原发性肺癌很困难，但也很重要，因为早期诊断可显著提高生存率。循环microRNA （miR）谱被认为是癌症（包括肺癌）的有前途的诊断和预后生物标志物。本研究的目的是通过测量循环miRs来建立早期检测原发性肺癌（NSCLC）的生物标志物。我们观察了配对的早期非小细胞肺癌患者和配对对照的血清和血浆样本。我们发现以下miRs的差异表达：miR-146b， miR-221, let-7a, miR-155, miR-17-5p， miR-27a和miR-106a，在NSCLC病例的血清中显著降低，而miR-29c显著升高。尽管我们没有观察到血浆中的任何显著差异，血清中的表达水平与血浆水平也没有很好的相关性，但这些组织类型是不同的，这可以解释结果的差异。此外，在所有患者中，血浆中let-7b表达的降低与更严重的癌症特异性死亡率有中度相关性，而在IA/B期患者中，血清中miR-223表达的降低与癌症特异性死亡率有中度相关性。此外，我们观察了参加国际辅助肺癌试验（IALT）的患者的福尔马林固定石蜡包埋肿瘤标本中生物学相关的miRs （miR-21, miR-29b, miR-34a/b/c， miR-155和let-7a）的表达，这是迄今为止对根治非小细胞肺癌患者进行辅助化疗的最大随机研究。目的是评估这些miRs的表达是否代表了生存的预后和预测价值。重要的是，某些miRs的表达与化疗耐药有关。然而，我们的结果表明，在这个大的患者队列中，检测的miRs表达模式既不能预测也不能预测预后。基于先前的NSCLC腺癌患者肺癌标本miR微阵列数据，我们研究了5个在肺癌中表达显著改变并与癌症特异性死亡率相关的miR的表达。其中三个miRs （miR-17， miR-21和miR-155）是潜在的致癌miRs，在肿瘤中表达增加，高表达水平与预后不良相关。我们研究了它们在来自马里兰州、挪威和日本的NSCLC患者的早期肺腺癌快速冷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miRs的表达升高与较差的癌症特异性死亡率相关。然而，只有miR-21在挪威队列中表现出相同的相关性，而在日本队列中表现出更差的无复发生存率。此外，与TNM I期患者相比，更晚期的肿瘤表达的miR-21水平明显更高，在TNM I期患者中，miR-21也与更差的癌症特异性死亡率和无复发生存率相关，独立于其他临床因素。我们发现，在来自世界不同地区的3个独立的NSCLC腺癌队列中，miR-21表达升高与较差的预后独立相关。这一发现具有翻译相关性；miR-21的表达作为NSCLC腺癌患者早期预后的生物标志物具有潜在的效用。虽然我们没有观察到FFPE组织中miR-21表达的显著增加，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，我们已经采取了系统生物学的方法来研究miRs在癌症中的作用。每个miR可以控制数百种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。我们证实或发现了在癌症中具有全面作用的mir: has-miR-103/106在癌症中下调，而has-miR-30变得最突出；has-miR-17/92家族在癌症中扩增，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。我们报道了第一个来自正常组织的miR网络，但也建立了耦合癌组织和非癌组织的miR网络，并确定了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，以生成癌症中全面的miR改变图谱。除了将miRs作为肺癌的重要生物标志物进行研究外，我们还研究了一个肿瘤抑制基因LKB1/STK11，该基因位于染色体19p13.3上，具有丝氨酸-苏氨酸激酶活性。染色体19p是吸烟者肺肿瘤中第二个最常见的缺失区域，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方法来评估原发性非小细胞肺癌的KLB1位点。我们的数据表明，在39%的测试样本中，LKB1基因通过纯合缺失（HD）或杂合性缺失（LOH）与体细胞突变完全失活，而在90%的NSCLC中，LKB1区域的HD或LOH导致染色体19p区域的失活。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，并假设雌激素受体和雌激素生物合成途径中的功能性单核苷酸多态性（snp）与较差的肺癌生存率相关。分析血清雌激素、孕酮、激素受体肿瘤信使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生存率较差。此外，雌激素受体α (er - α)多态性变异等位基因（rs2228480）在所有队列中与肿瘤er - α水平升高和生存率降低显著相关。结果与性别和激素替代治疗无关。因此，我们报道了在男性和女性肺癌患者中，血清雌激素升高与较差的生存率显著相关。此外，我们研究了一个非常重要的机制，细胞永生，这是癌症的标志之一，依赖于端粒长度维持机制（TMM）的活性，端粒酶或端粒的选择性延长（ALT）。我们检查了43例恶性胸膜间皮瘤（MPMs）。TMMs被广泛认为是癌症治疗的潜在靶点，端粒酶抑制剂已经进入临床试验。我们询问有多少比例的MPMs使用ALT和/或端粒酶。我们发现43例MPMs中有43例端粒酶阳性[+]和alt阴性[-]。为了研究MPM细胞是否对端粒酶的激活异常敏感，我们在细胞永生的体外模型中检测了tmm的激活，在该模型中，正常的胸膜间皮细胞被SV40转导。我们发现胸膜间皮瘤细胞能够在体外激活两种TMM，并且观察到100%的MPMs是端粒酶[+]，这表明在这种肿瘤类型的肿瘤发生过程中存在体内因素选择端粒酶活性。我们的结论是，MPM是一种可以考虑抗端粒酶治疗的肿瘤。在我们的研究中描述的基因和机制对肺癌的发生、进展和结局很重要，是肺癌的潜在分子预后和诊断生物标志物。

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.