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Adenocarcinoma is the most common type of lung cancer, the leading cause of cancer deaths in the world. Early detection is the key to improve the survival of lung adenocarcinoma patients. We have previously shown that microRNAs (miRNAs) were stably present in sputum and could be applied to diagnosis of lung cancer. The aim of our study was to develop a panel of miRNAs that can be used as highly sensitive and specific sputum markers for early detection of lung adenocarcinoma. Our study contained 3 phases: (i) marker discovery using miRNA profiling on paired normal and tumor lung tissues from 20 patients with lung adenocarcinoma; (ii) marker optimization by real-time reverse transcription-quantitative polymerase chain reaction on sputum of a case–control cohort consisting of 36 cancer patients and 36 health individuals and (iii) validation on an independent set of 64 lung cancer patients and 58 cancer-free subjects. From the surgical tissues, 7 miRNAs with significantly altered expression were identified, of which “4” were overexpressed and “3” were underexpressed in all 20 tumors. On the sputum samples of the case–control cohort, 4 (miR-21, miR-486, miR-375 and miR-200b) of the 7 miRNAs were selected, which in combination produced the best prediction in distinguishing lung adenocarcinoma patients from normal subjects with 80.6% sensitivity and 91.7% specificity. Validation of the marker panel in the independent populations confirmed the sensitivity and specificity that provided a significant improvement over any single one alone. The sputum markers demonstrated the potential of translation to laboratory settings for improving the early detection of lung adenocarcinoma.
Non–small-cell lung cancer (NSCLC) is the leading cause of cancer death in the United States. NSCLC is histologically subdivided into 4 major subtypes with distinct pathological characteristics: adenocarcinoma, squamous cell carcinoma, large cell carcinoma and “other” (neuroendocrine cancers, carcinoids, etc.). The disease is usually diagnosed at advanced stages when the prognosis is poor, resulting in an overall 5-year survival rate of ∼14%.1 However, the 5-year survival rate in patients with Stage I NSCLC that has been resected can be as high as 83%.1 Therefore, finding early stage NSCLC may reduce the mortality.1 In particular, early identification of lung adenocarcinoma is clinically important because it is now the most common type of lung cancer,1 accounting for 40% of all NSCLCs. Furthermore, the incidence of lung adenocarcinoma is on the rise in many countries, mainly in women and nonsmokers.2, 3 In addition, because adenocarcinoma arises in peripheral lung tissue and originates from the smaller airways, it is more difficult to be detected by bronchoscopy or sputum cytology.3 Moreover, computed tomography (CT) provides excellent anatomic information and can detect lung tumor at small size; however, the improved sensitivity is associated with over diagnosis.1–3 Thus, the major obstacle in management of lung adenocarcinoma is the lack of adequate method for its early detection.
MicroRNAs (miRNAs) are a new class of small noncoding RNAs that regulate gene expression and are involved in a variety of biologic and pathologic processes.4 The differential expression of miRNAs in human cancers and its potential diagnostic values have been previously investigated.4–7 For instance, by analyzing changes of a large-scale miRNAs on 540 human cancer specimens including lung, breast, stomach, prostate, colon and pancreatic tumors, Volinia et al. identified a solid cancer miRNA signature composed of a large portion of overexpressed miRNAs that provides potential diagnostic targets for the tumors.4 Our recent proof of principle study8 showed that endogenous miRNAs were present in sputum in a remarkably stable form and could reliably be detected by real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Furthermore, detecting elevated expression of a single miRNA, miR-21, produced a higher sensitivity in diagnosis of lung cancer compared to sputum cytology. Our data suggested that the measurement of altered miRNA expressions in sputum sample could be a useful noninvasive approach for lung cancer diagnosis. However, the sensitivity reached by a single miRNA is low for clinical application.8
It has been widely accepted that lung tumor is a heterogeneous disease and develops from complex and multistep processes.2, 9 We, therefore, hypothesized that simultaneous assessment of a panel of tumor-specific miRNAs that, used in combination in sputum, could provide a highly sensitive and specific diagnostic test for early stage lung adenocarcinoma. To verify the hypothesis, we first identified miRNA signatures of Stage I lung adenocarcinoma using miRNA profiling on primary tumor tissues. From these signatures, we then optimized and validated a panel of miRNAs that could be detected in sputum for the early detection of lung adenocarcinoma.
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The development of highly accurate biomarkers that can be detected in easily accessible body fluids is a major research effort in the field of lung cancer early detection.17 Sputum, particularly, has been considered as potential surrogate material for noninvasive diagnosis of lung cancer, because it is a mirror to lung disease.17 Conventional cytologic analysis of sputum has been used clinically to diagnose lung cancer; however, it was no more effective than chest radiographs in detecting lung cancer in several large prospective randomized trials.18 The molecular genetic alterations could occur before morphological changes that can be found by a cytological test.8, 11, 12, 19–22 Furthermore, the molecular genetic changes seen in sputum may reflect the same abnormalities found in lung tumors.11, 21 Therefore, there is a long history of identifying and developing molecular genetic changes that can be tested in sputum as biomarkers.8, 11, 12, 17, 19–22 For instance, mutations of oncogene (e.g., K-ras) or tumor suppressor gene (e.g., P53) were detected in sputum of patients with primary adenocarcinoma of the lung.19, 20 Hypermethylation of p16 gene was found in sputum collected from patients with lung cancer, 5–35 months before sputum cytological and clinical diagnoses.21 However, to date there is no molecular genetic marker accepted in clinical settings.
In our recent proof of principle study,8 we demonstrated that measuring elevated expression level of a single miRNA in sputum produced higher sensitivity in identification of lung cancer than did conventional sputum cytology. To enhance the diagnostic power of miRNAs in sputum for lung adenocarcinoma, here we developed and characterized a sputum-based miRNA marker panel with a sensitivity of 80.6% and a specificity of 91.7%. Our study further extends our previous research efforts to develop sputum-based diagnostic tool for lung cancer.8, 11, 12, 22 Given the expenses associated with quantitative molecular analyses, a marker panel with the smallest number of miRNAs and highest diagnostic accuracy would provide a cost-effective diagnostic assay for lung adenocarcinoma.
Among the 4 miRNAs identified, upregulation of miR-21 has been found in many human cancer specimens.23 Therefore, extensive efforts have been taken to identify the downstream genes and gene networks regulated by miR-21 and the upstream factors that can regulate dysfunction of miR-21.23–25 For example, elevated miR-21 expression might be associated with apoptosis inhibition and acquisition of invasive properties, likely mediated by its downregulating effects on the expression of target tumor suppressors PTEN, TPM and PDCD4.23–25 More importantly, miR-21 itself displays oncogenic activity and can be classed as an oncomir, whose overexpression lead to tumor development and progression.24 This study confirmed our previous finding8 that the assessment of miR-21 overexpression in sputum had higher sensitivity compared to cytologic examination. Therefore, miR-21 can serve as an important biomarker for the early detection of lung cancer. MiR-200b locates on chromosome 1p36.33, one of the most common regions with genomic amplicons in solid tumors including lung cancer.26–29 Although biological mechanism of miR-200b dysfunction in lung tumorigenesis is unclear, miR-200b was recently identified as one of a set of miRNAs whose aberrant expressions were related to recurrence of Stage I NSCLC after surgical resection.30 Consistently, we herein found that miR-200b overexpression existed in lung adenocarcinomas, one of the major histological types of NSCLC. Altogether, the observations suggest that miR-200b could be a potential target of the genomic amplification in 1p36.33 and its activation might be involved in lung carcinogenesis. MiR-375 downregulation was found in some human malignancies.31, 32 However, miR-375 was consistently upregulated in adenocarcinoma rather than squamous cell carcinoma of lungs.33 Furthermore, when comparing cancerous tissue expression between adenocarcinoma and squamous cell carcinoma patients with esophagus cancer, Mathé et al.32 found that miR-375 was elevated in adenocarcinoma patients. In good agreement with the findings, our present data showed that miR-375 was overexpressed in lung adenocarcinoma, and moreover, measuring its expression in sputum displayed higher accuracy in diagnosis of lung adenocarcinoma compared to squamous cell lung cancer. On the other hand, miR-486 is located on one of the most frequent genomic rearrangement regions, chromosome 8p11.21 that contain potential tumor suppressor genes in lung tumorigenesis.26, 27 Navon et al. recently found that miR-486 was underexpressed in 8 types of human tumors including lung cancer.34 The observation from our present research is consistent with the previous findings, suggesting that miR-486 might be a potential tumor suppressor in carcinogenesis. Although miR-182 was not eventually included in the panel of the 4 miRNAs, its overexpression was found in primary NSCLC tissues and sputum from the patients. MiR-182 is a member of a miRNA cluster in a chromosomal locus (7q31-34) frequently amplified in solid tumors.35 Segura et al. recently found that miR-182 was commonly upregulated in human melanoma cell lines and tissue samples and this upregulation correlated with gene copy number in a subset of melanoma cell lines.35 Furthermore, the aberrant miR-182 expression could promote tumorigenesis by repressing FOXO3 and microphthalmia-associated transcription factor in several types of human cancers.36–38 Our primary goal of this study is marker development. We showed for the first time that measuring altered expressions of a small panel of miRNAs in sputum might be a potential noninvasive test for the early detection of lung adenocarcinoma. The biological relevance of the miRNA dysfunctions in lung tumorigenesis are currently being investigated at our laboratory.
The panel of sputum markers could also identify squamous cell lung cancer with 64.1% sensitivity and 71.3% specificity. However, the markers are more accurate to lung adenocarcinoma with 80.6% sensitivity and 92.5% specificity. The main reason might be that the miRNAs are identified from surgically resected primary adenocarcinomas, thus should be more specific for the type of NSCLC. It is also not surprising to find that the abnormal miRNAs expression levels are related to the tumors located in peripheral airways, because most peripheral tumors are adenocarcinomas. These observations would be clinically important, because the majority of NSCLCs detected by cytologic analysis and visible by bronchoscopy are squamous cell carcinoma predominantly locating in central areas of the lungs rather than adenocarcinoma that is the most common type in NSCLC. Once confirmed, the sputum miRNA panel might improve the detection rate for lung adenocarcinomas that are more difficult to be found by these conventional techniques.
Most of the previously identified lung cancer–associated molecular genetic changes were related to the smoking status. Some of the changes can be found in healthy smokers who never develop lung cancer.2, 3, 8, 17 The use of such molecular genetic alterations as biomarkers might produce high false positive diagnostic rate or over diagnosis, thus impeding their application in clinical settings in screening or early detection of lung cancer. The 4 miRNA markers identified from the present research is encouraging, because expression levels of the miRNAs appear to be independent of subject age, gender, ethnic subgroup and tobacco smoking. The identified miRNAs could dysregulate in a cancer-specific manner. In addition, no significant differences of the miRNA expression levels were observed for the cancerous samples at different stages of the disease, implying that the potential markers were not stage-specific. The results further provide evidence that this miRNA marker panel might be useful in the early detection of lung adenocarcinoma, although whether the expression levels of the 4 miRNAs are affected in noncancer-associated lung pathologies remains to be investigated.
This panel of miRNA markers detected by RT-qPCR platform provides a significant improvement over any single one and hence, shows promise as a lung adenocarcinoma–specific test on sputum. In the future, developing and using an independent methodology, for example, solution hybridization,39 to evaluate the expression levels of the miRNAs may continue to improve efficiency of the sputum-based biomarkers. Furthermore, comparing the miRNAs on sputum to CT imaging for the early detection of lung adenocarcinoma will lead to more understanding the diagnostic value of the biomarkers. In addition, integration of the sputum-based markers with the current conventional modalities, especially CT, could facilitate noninvasive diagnostic efficiency and accuracy for early lung adenocarcinoma or screening of high-risk patients for the cancer.
In conclusion, we have developed a panel of miRNAs that can be reliably measured in sputum. Detection of the miRNAs could be used as a noninvasive and cost-effective diagnostic tool for early lung adenocarcinoma. Nonetheless, a large multicenter clinical project to further validate the full utility is warranted before it could potentially be adopted in routine clinical settings.