Implications of miRNAs in the Diagnosis of Lung Cancer

Identifying patients with early-stage lung cancer who will benefit the most from effective therapies may reduce the mortality of this deadly disease. Early detection is thus a key to improving the survival of patients with lung cancer. The results of investigational studies suggest that miRNAs may become promising biomarkers for risk assessment and diagnosis of lung cancer ( Tables 15.1 and 15.2 ).

The let-7 family is a global genetic regulator important in controlling the expression of lung cancer oncogenes. Chin et al. sequenced the let-7 complementary sites (LCS) in the Kirsten rat sarcoma ( KRAS ) 3ʹ UTR from 74 cases of NSCLC and identified a SNP at LCS6 significantly associated with an increased risk for NSCLC among moderate smokers (odds ratio, 2.3; 95% CI, 1.1–4.6). The LCS6 variant allele showed a 2.3-fold increase in risk for NSCLC among patients who smoked the equivalent of less than 40 pack-years.

A survey has reported that the SNP rs11614913 in miR-196a2 may affect mature miR-196a expression and target mRNA-binding activity and is significantly associated with survival from NSCLC. In a case–control study of 1058 patients with incident lung cancer and 1035 cancer-free controls in a Chinese population, Tian et al. found that miR-196a2 rs11614913 variant homozygote CC was associated with a significant increase of approximately 25% (odds ratio, 1.25; 95% CI, 1.01–1.54) in the risk of lung cancer compared with the wild-type homozygote TT and heterozygote TC. To further determine whether any association exists between four common SNPs (miR-196a2 C>T, rs11614913; miR-146a G>C, rs2910164; miR-499 A>G, rs3746444; and miR-149 C>T, rs2292832) and the risk for lung cancer, He et al. performed a meta-analysis of 40 published case–control studies. Their results demonstrated that the rs11614913 TT genotype was significantly associated with a decreased risk of lung cancer for an Asian population subgroup (TT vs. CC: odds ratio, 0.7; 95% CI, 0.57–0.85; p = 0.284). Squamous cell carcinoma is one major subtype of lung cancer for which biomarkers are urgently needed to aid patient management. Measuring the miRNA expression in cancerous and noncancerous tissue pairs collected from 60 Chinese patients with squamous cell carcinoma (stages I–III), Tan et al. identified a panel of five miRNAs (miR-30a, miR-140-3p, miR-182, miR-210, and miR-486-5p) that distinguished squamous cell carcinoma from normal lung tissues with an accuracy of 94%. They also showed that high expression of miR-31 was associated with poor survival among patients with squamous cell carcinoma.

Recent advances in the treatment of lung cancer require greater accuracy in the subclassification of NSCLC. Using a high-throughput microarray to measure the miRNA expression levels in 122 samples of adenocarcinoma and squamous cell carcinoma, Lebanony et al. identified miR-205 as a highly specific marker (96% sensitivity and 90% specificity) for squamous cell carcinoma. This standardized diagnostic assay may provide accurate subclassification of NSCLC. Fassina et al. investigated the accuracy of miRNAs in differentiating squamous cell carcinoma from adenocarcinoma within scant and distorted specimens obtained by transthoracic needle aspiration. Quantification of the let-7 family and miR-205 expression levels in 18 adenocarcinoma and 13 squamous cell carcinoma specimens by quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed a significant upregulation of the let-7 family and a significant downregulation of miR-205 in adenocarcinoma specimens (all, p < 0.05). Xing et al. profiled miRNA expression signatures in 15 samples of lung squamous cell carcinoma and matched normal lung samples with an miRNA array (GeneChip; Affymetrix, Santa Clara, CA, USA). They identified three miRNAs (miR-205, miR-210, and miR-708) that distinguished the sputum samples of patients with stage I lung squamous cell carcinoma from those of healthy individuals with 73% sensitivity and 96% specificity. Early detection is also the key to improving the survival of patients with lung adenocarcinoma. Using miRNA profiling on 20 paired samples of adenocarcinoma and normal lung tissue, Yu et al. identified four miRNAs (miR-21, miR-200b, miR-375, and miR-486) that distinguished the sputum samples of patients with stage I lung adenocarcinoma from those of healthy individuals with 81% sensitivity and 92% specificity.

Other studies were designed to identify serum-based miRNAs with the ability to diagnose NSCLC at an early stage. Owing to their high stability during storage and handling, miRNAs are optimal biomarkers present in the blood, urine, and other body fluids. Foss et al. performed miRNA profiling on total RNA extracted from serum obtained from 11 patients with early-stage NSCLC and 11 controls. The authors found that the expression of miR-574-5p and miR-1254 was significantly increased in the samples of early-stage NSCLC with respect to the controls ( p = 0.0277). Receiver operating characteristic curves plotting these two miRNAs were able to discriminate early-stage NSCLC from control samples with 82% sensitivity and 77% specificity. Using quantitative RT-PCR to measure the circulating levels of miRNAs in paired serum and plasma samples from 220 patients with early-stage NSCLC and 220 matched controls, Heegaard et al. also demonstrated that the expression levels of let-7a, miR-17-5p, miR-27a, miR-106a, miR-146b, miR-155, and miR-221 were significantly reduced in the serum of patients with NSCLC, whereas miR-29c was significantly increased (all, p < 0.05). Performing risk–score analysis to evaluate the diagnostic values of serum miRNA profiling among 400 patients with NSCLC and 200 controls, Chen et al. showed that a panel of 10 serum miRNAs (miR-20a, miR-24, miR-25, miR-145, miR-152, miR-199a-5p, miR-221, miR-222, miR-223, and miR-320) accurately distinguished patients with NSCLC from controls even up to 33 months before the clinical diagnosis of NSCLC.

The levels of miRNAs in serum are stable, reproducible, and consistent among individuals of the same species. Chen et al. used Solexa sequencing on serum miRNAs of human participants and obtained two miRNAs (miR-25 and miR-223) specific for NSCLC. The study conducted expression profiles of serum miRNAs in 21 healthy human patients and 11 NSCLC patients. Their results were validated in an independent cohort of 152 lung cancer patients and 75 healthy controls. The results of these analyses suggest that the copy numbers of these two serum miRNAs may serve as biomarkers for the detection of NSCLC. Cazzoli et al. used exosome-based techniques to analyze the miRNAs of 30 plasma samples (10 lung adenocarcinomas, 10 lung granulomas, and 10 healthy smokers) and subsequently validated them on an independent group of 105 specimens. The results showed that a screening test of four miRNAs (miR-139-5p, miR-200b-5p, miR-378a, and miR-379) was useful to divide the nodule and nonnodule groups (97.5% sensitivity, 72% specificity, and 90.8% area under the receiver operating characteristic curve [AUC]). The authors also developed a diagnostic test of six miRNAs (miR-30a-3p, miR-100, miR-151a-5p, miR-154-3p, miR-200b-5p, and miR-629) to discriminate between lung adenocarcinoma and granuloma (96% sensitivity, 60% specificity, and 76% AUC).

To examine whether circulating miRNAs have the potential to become suitable blood-based markers for the diagnosis and progression of lung cancer, Roth et al. measured the concentrations of four miRNAs in the serum of 35 patients with lung cancer and seven patients with benign lung tumors. The levels of miR-10b ( p = 0.002) and miR-141 ( p = 0.0001) were significantly higher in patients with lung cancer than in patients with benign disease. High serum concentrations of miR-10b were also found to be associated with lymph node metastasis among patients with lung cancer. Circulating cell-free miRNAs in pleural effusion are also potential biomarkers for cancer. Using microarrays to screen miRNAs in 10 malignant pleural effusions associated with lung adenocarcinoma and 10 benign pleural effusions, Han et al. showed that miR-198 was significantly downregulated in malignant pleural effusion compared with benign pleural effusion ( p = 0.002). The results of miRNA microarray analysis were confirmed by quantitative RT-PCR using a validation set comprising 45 malignant pleural effusions associated with lung adenocarcinoma and 42 benign pleural effusions. The AUC for miR-198 was 0.887.

Although early detection using chest x-ray and spiral computed tomography (CT) has resulted in a marked increase in the number of lung cancer diagnoses, these efforts may also lead to unnecessary treatments, and this possibility indicates the need for biomarkers of aggressive disease. Boeri et al. explored the miRNA expression profiles of 74 plasma samples collected during the 5-year screening plan and identified 12 of 15 samples collected before lung cancer detection by spiral CT, with sensitivity of 80% and specificity of 90%. The 5-year screening plan was a longitudinal analysis of 3246 current or former smokers screened for lung cancer beginning in 1998 either in the United States or in Italy to assess whether CT screening might increase the frequency of lung cancer diagnosis. The median amount of follow-up from the initial CT evaluation to the mortality end point was nearly 5 years. The most frequently deregulated miRNAs were miR-28-3p, miR-30c, miR-92a, miR-140-5p, miR-451, and miR-660.

Brain metastasis affects approximately 25% of patients with NSCLC during their lifetime. Arora et al. performed miRNA microarray profiling on samples from seven patients with NSCLC with brain metastasis and six without brain metastasis, and confirmed that the expression of miR-328 was able to correctly classify patients with and without brain metastasis. This miRNA may be incorporated into clinical treatment decision making to stratify patients with NSCLC who have a higher risk for brain metastasis. Profiling miRNAs extracted from 527 stage I NSCLCs on the human miRNA expression profiling panel, Lu et al. found that miR-145∗ was associated with brain metastasis by virtue of inhibiting cell invasion and metastasis. This miRNA holds potential as a target for preventing and treating brain metastasis among patients with stage I NSCLC.

MicroRNAs as Prognostic Biomarkers for Lung Cancer

Despite the availability of effective treatments, recurrence is common even for early-stage lung cancer. Prognostic biomarkers that can predict tumor progression and survival are required to provide better guidance on postoperative surveillance and therapeutic decisions for patients with lung cancer. Recent evidence suggests that specific miRNAs with altered expression levels have great potential to serve as prognostic biomarkers for lung cancer ( Tables 15.3 and 15.4 ).

TABLE 15.3

Single MicroRNAs (miRNAs) as Prognostic Biomarkers for Lung Cancer

miRNAs	Deregulation in Cancer	Materials	Descriptions	References
let-7	Downregulated	Tissue	Overexpression of let-7 in NSCLC cells inhibits cell growth in vitro
let-7a-2	Downregulated	Tissue	Low expression of let-7a-2 correlates with poor survival of patients with lung adenocarcinoma
let-7f	Upregulated	Plasma	Expression level of let-7f is associated with overall survival in NSCLC
miR-16	Downregulated	Serum	High expression of miR-16 is associated with significantly better survival in patients with advanced NSCLC
miR-21	Upregulated	Tissue	Overexpression of miR-21 is an independent negative prognostic factor for overall survival in NSCLC Inhibition of miR-21 inhibits cell growth in vitro and inhibits tumor growth in xenograft mouse models of lung adenocarcinoma High expression of miR-21 has a negative impact on relapse-free survival in lung adenocarcinoma and overall survival in NSCLC
miR-30c-1	Downregulated	Tissue	The expression of the host nuclear transcription factor Y gene is correlated with pri-miR-30c-1, but not with rs928508 genotypes among patients with NSCLC
miR-30e-3p	Downregulated	Plasma	The expression level of miR-30e-3p is associated with short disease-free survival in NSCLC
miR-31	Upregulated	Tissue	miR-31 represses DICER1 activity but not PPP2R2A or LATS2 in lung squamous cell carcinoma In vitro functional assays show that miR-31 increases cell migration, invasion, and proliferation in an ERK1/2 signaling-dependent manner in lung adenocarcinoma
miR-34a	Downregulated	Tissue	A relation has been found between MIRN34A methylation and miR-34a expression in NSCLC
miR-34b	Downregulated	Tissue	Overexpression of miR-34b decreases the expression of c-Met in NSCLC cells
miR-34b/c	Downregulated	Tissue	Ectopic expression of miR-34b/c in lung adenocarcinoma cells decreases cell proliferation, migration, and invasion
miR-145	Downregulated	Tissue	miR-145 regulates SOX2 and OCT4 translation, and p53 regulates miR-145 expression
miR-146b	Upregulated	Tissue	High expression of miR-146b correlates with poor overall survival for patients with lung squamous cell carcinoma
miR-149	Downregulated	Tissue	miR-149 may be involved in the pathogenesis of NSCLC
miR-155	Upregulated	Tissue	High expression of miR-155 correlates with poor survival of patients with lung adenocarcinoma High expression of miR-155 correlates with poor overall survival for patients with lung squamous cell carcinoma
miR-186	Downregulated	Cell line	Cyclin D1, CDK2, and CDK6 are each directly inhibited by miR-186, and restoring their expression levels reverses miR-186-mediated inhibition of cell cycle progression
miR-196a	Upregulated	Tissue	miR-196a may be involved in the pathogenesis of NSCLC
miR-196a2	Upregulated	Tissue	rs11614913 CC is associated with a significant increase in mature miR-196a expression, but not with changes in levels of the precursor for NSCLC
miR-367	Upregulated	Tissue	SOX2 and OCT4 transcription factors regulate the expression of miR-367
miR-651	Downregulated	Blood	The FAS:rs2234978 G allele is significantly associated with survival in early-stage NSCLC, and the FAS single nucleotide polymorphism created a miR-651 functional binding site
miR-708	Upregulated	Tissue	Forced miR-708 expression reduces TMEM88 transcript levels and increases the rate of cell proliferation, invasion, and migration in culture

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