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Aim: We externally validate plasma miRNAs biomarkers for lung cancer in a large and retrospective sample set collected from a geographically distant population. Methods: Plasma samples are tested blindly to the clinical annotations by using PCR for quantitation of the four miRNAs in cohort 1 consisting of 232 lung cancer cases and 243 controls and cohort 2 comprising 239 cases and 246 controls. Results: Combined use of the four plasma miRNAs has 91% sensitivity and 95% specificity for diagnosis of lung cancer, and 85% sensitivity for early-stage lung cancer, while maintaining a specificity of 95%. Conclusion: The diagnostic values of the biomarkers are reproducibly confirmed in the independent and large sample sets, providing an assay for lung cancer detection.

Lung cancer is primarily caused by cigarette smoking and the leading cancer killer in the USA and across the world. Early detection of lung cancer by low-dose CT (LDCT) can reduce the mortality. However, LDCT dramatically increases the number of indeterminate pulmonary nodules (PNs), leading to overdiagnosis. Having a definitive preoperative diagnosis of malignant PNs is clinically important. Using microarray and droplet digital PCR to directly profile plasma miRNA expressions of 135 patients with PNs, we identified 11 plasma miRNAs that displayed a significant difference between patients with malignant versus benign PNs. Using multivariate logistic regression analysis of the molecular results and clinical/radiological characteristics, we developed an integrated classifier comprising two miRNA biomarkers and one radiological characteristic for distinguishing malignant from benign PNs. The classifier had 89.9% sensitivity and 90.9% specificity, being significantly higher compared with the biomarkers or clinical/radiological characteristics alone (all p < 0.05). The classifier was validated in two independent sets of patients. We have for the first time shown that the integration of plasma biomarkers and radiological characteristics could more accurately identify lung cancer among indeterminate PNs. Future use of the classifier could spare individuals with benign growths from the harmful diagnostic procedures, while allowing effective treatments to be immediately initiated for lung cancer, thereby reduces the mortality and cost. Nevertheless, further prospective validation of this classifier is warranted., (© 2017 UICC.)

Background: Abnormal microRNA (miRNA) expressions and promoter methylation of genes detected in sputum may provide biomarkers for non-small lung cancer (NSCLC). Here, we evaluate the individual and combined analysis of the two classes of sputum molecular biomarkers for NSCLC detection., Results: We analyze expression of 3 miRNAs (miR-21, miR-31, and miR-210) and methylation of 3 genes ( RASSF1A , PRDM14 , and 3OST2 ), which were previously identified as potential biomarkers for NSCLC, in sputum of a set of 117 stage I NSCLC patients and 174 cancer-free smokers. The results are validated in a different set of 144 stage I NSCLC patients and 171 controls. The panel of 3 miRNA biomarkers has 81.5 % sensitivity and 85.9 % specificity; the panel of 3 methylation biomarkers displays 82.9 % sensitivity and 76.4 % specificity for NSCLC detection. Integrated analysis of 2 miRNAs (miR-31 and miR-210) and 2 genes ( RASSF1A and 3OST2 ) yields higher sensitivity (87.3 %) and specificity (90.3 %) compared with the individual panels of the biomarkers ( P  < 0.05). Combined analysis of all the 3 miRNAs and 3 genes does not have performance superior to that of the panel of 2 miRNAs and 2 genes ( P  > 0.05). The performance of combined use of the two classes of biomarkers was confirmed in the validation set., Conclusions: The integration of two different classes of biomarkers synergistically improves both the sensitivity and the specificity for the early detection of NSCLC.

Molecular analysis of sputum presents a noninvasive approach for diagnosis of lung cancer. We have shown that dysregulation of small nucleolar RNAs (snoRNAs) plays a vital role in lung tumorigenesis. We have also identified six snoRNAs whose changes are associated with lung cancer. Here we investigated if analysis of the snoRNAs in sputum could provide a potential tool for diagnosis of lung cancer. Using qRT-PCR, we determined expressions of the six snoRNAs in sputum of a training set of 59 lung cancer patients and 61 cancer-free smokers to develop a biomarker panel, which was validated in a testing set of 67 lung cancer patients and 69 cancer-free smokers for the diagnostic performance. The snoRNAs were robustly measurable in sputum. In the training set, a panel of two snoRNA biomarkers (snoRD66 and snoRD78) was developed, producing 74.58% sensitivity and 83.61% specificity for identifying lung cancer. The snoRNA biomarkers had a significantly higher sensitivity (74.58%) compared with sputum cytology (45.76%) (P < 0.05). The changes of the snoRNAs were not associated with stage and histology of lung cancer (All P >0.05). The performance of the biomarker panel was confirmed in the testing cohort. We report for the first time that sputum snoRNA biomarkers might be useful to improve diagnosis of lung cancer.

Purpose: MicroRNAs (miRNAs) play important roles in the initiation and progression of lung cancer. Measuring miRNA expression levels in sputum could provide a potential approach for the diagnosis of lung cancer. The emerging digital PCR is a straightforward technique for precise, direct, and absolute quantification of nucleic acids. The objective of the study was to investigate whether digital PCR could be used to quantify miRNAs in sputum for lung cancer diagnosis., Methods: We first determined and compared dynamic ranges of digital PCR and conventional quantitative reverse transcriptase PCR (qRT-PCR) for miRNA quantification using RNA isolated from sputum of five healthy individuals. We then used digital PCR to quantify copy number of two lung cancer-associated miRNAs (miR-31 and miR-210) in 35 lung cancer patients and 40 cancer-free controls., Results: Copy number of the miRNAs measured by digital PCR displayed a linear response to input cDNA amount in a twofold dilution series over seven orders of magnitude. miRNA quantification determined by digital PCR assay was in good agreement with that obtained from qRT-PCR analysis in sputum. Furthermore, combined quantification of miR-31 and miR-210 copy number by using digital PCR in sputum of the cases and controls provided 65.71 % sensitivity and 85.00 % specificity for lung cancer diagnosis., Conclusion: As digital PCR becomes more established, it would be a robust tool for quantitative assessment of miRNA copy number in sputum for lung cancer diagnosis.

Introduction: Computed tomography (CT) plays a central role in lung cancer diagnosis. However, CT has relatively low specificity, presenting a challenge in clinical settings. We previously identified 12 microRNAs (miRNAs) whose expressions in tumor tissues were associated with lung cancer., Methods: Using quantitative reverse transcriptase polymerase chain reaction, we aimed to identify miRNA biomarkers in sputum that could complement CT for diagnosis of lung cancer., Results: In a training set consisting of 66 lung cancer patients and 68 cancer-free smokers, 10 of the 12 miRNAs were differentially expressed between the cases and controls (p ≤ 0.01). From the miRNAs, a logistic regression model was built on the basis of miR-31 and miR-210, both of which had the best prediction for lung cancer, producing an area under receiver operating characteristic curve of 0.83. Combined use of the two miRNAs yielded 65.2% sensitivity and 89.7% specificity, CT had 93.9% sensitivity and 83.8% specificity for lung cancer diagnosis. Notably, combined analysis of the miRNA biomarkers and CT produced a higher specificity than does CT used alone (91.2% versus 83.8%; p < 0.05). The diagnostic performance of the biomarkers was confirmed in a testing set comprising 64 lung cancer patients and 73 cancer-free smokers., Conclusion: The sputum miRNA biomarkers might be useful in improving CT for diagnosis of lung cancer, but further independent validation on an external and prospective cohort of patients is required.

Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related death. Developing minimally invasive techniques that can diagnose NSCLC, particularly at an early stage, may improve its outcome. Using microarray platforms, we previously identified 12 microRNAs (miRNAs) the aberrant expressions of which in primary lung tumors are associated with early-stage NSCLC. Here, we extend our previous research by investigating whether the miRNAs could be used as potential plasma biomarkers for NSCLC. We initially validated expressions of the miRNAs in paired lung tumor tissues and plasma specimens from 28 stage I NSCLC patients by real-time quantitative reverse transcription PCR, and then evaluated diagnostic value of the plasma miRNAs in a cohort of 58 NSCLC patients and 29 healthy individuals. The altered miRNA expressions were reproducibly confirmed in the tumor tissues. The miRNAs were stably present and reliably measurable in plasma. Of the 12 miRNAs, five displayed significant concordance of the expression levels in plasma and the corresponding tumor tissues (all r>0.850, all P<0.05). A logistic regression model with the best prediction was defined on the basis of the four genes (miRNA-21, -126, -210, and 486-5p), yielding 86.22% sensitivity and 96.55% specificity in distinguishing NSCLC patients from the healthy controls. Furthermore, the panel of miRNAs produced 73.33% sensitivity and 96.55% specificity in identifying stage I NSCLC patients. In addition, the genes have higher sensitivity (91.67%) in diagnosis of lung adenocarcinomas compared with squamous cell carcinomas (82.35%) (P<0.05). Altered expressions of the miRNAs in plasma would provide potential blood-based biomarkers for NSCLC.

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., (Copyright © 2010 UICC.)

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death. Early detection of NSCLC will improve its outcome. We previously identified genetic signatures whose genomic copy number aberrations were associated with early stage NSCLC. Here, we aimed to develop a panel of genes that could be detected in sputum for NSCLC early detection. We first optimized a panel of genes by using an in situ minichip for measuring changes of the signatures in sputum of a case-control cohort of 49 NSCLC patients, 49 patients with chronic obstructive pulmonary disease (COPD), and 49 healthy smokers. We then validated the genes in an independent cohort of 69 NSCLC patients and 65 noncancer subjects. The results were compared with those of sputum cytology. Fifteen genes showed significant differences of their copy number changes in sputum between NSCLC and both COPD and healthy subjects. A logistic regression model with the best prediction was built on the basis of 6 genes, ENO1, FHIT, HYAL2, SKP2, p16, and 14-3-3zeta. The composite of the 6 genes produced 86.7% sensitivity and 93.9% specificity in distinguishing stage I NSCLC patients from the noncancer individuals. Furthermore, the genes had higher sensitivity (86.9%) in identification of squamous cell carcinoma (SCC) than in adenocarcinoma of the lungs (80.8%; P < 0.05). Validation of the genes in the independent cohort confirmed their diagnostic power that also showed higher accuracy for lung SCCs than for sputum cytology. The gene panel could provide sputum-based markers that have the potential to improve early detection of lung SCCs., (©2010 AACR.)

Squamous cell carcinoma is a common form of lung cancer, the leading cause of cancer deaths in the world. Identifying early stage lung squamous cell carcinoma patients who would benefit most from effective therapies will reduce the mortality. We have previously shown that microRNAs (miRNAs) were stably present in sputum and potentially useful in diagnosis of lung cancer. The objective of this study was to develop a panel of miRNAs that can be used as a sputum-based test for early stage squamous cell carcinoma of the lungs. This study contained three phases: (1) marker discovery by profiling miRNA expression signatures on 15 lung squamous cell carcinoma and matched normal lung tissue samples with GeneChip miRNA Array; (2) marker optimization by real-time quantitative RT-PCR on sputum of a case-control cohort of 48 stage I lung squamous cell carcinoma patients and 48 healthy individuals; and (3) marker validation on an independent set including 67 lung squamous cell carcinoma patients and 55 healthy subjects. On the surgical tissues, six miRNAs were identified, of which three were overexpressed and three were underexpressed in all 15 tumors. On the sputum samples of the case-control cohort, three (miR-205, miR-210 and miR-708) of the six miRNAs were selected, which in combination produced the best prediction in distinguishing lung squamous cell carcinoma patients from normal subjects with 73% sensitivity and 96% 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 showed the potential to improve the early detection of lung squamous cell carcinomas.

Unlabelled: Analysis of molecular genetic markers in biological fluids has been proposed as a useful tool for cancer diagnosis. MicroRNAs (miRNAs) are small regulatory RNAs that are frequently dysregulated in lung cancer and have shown promise as tissue-based markers for its prognostication. The aim of this study was to determine whether aberrant miRNA expression can be used as a marker in sputum specimen for the diagnosis of non-small cell lung cancer (NSCLC)., Experimental Design: expressions of mature miRNAs, mir-21 and mir-155, were examined by real-time reverse transcription polymerase chain reaction (RT-PCR) and normalized to that of control miRNA, U6B, in sputum of 23 patients with NSCLC and 17 cancer-free subjects. The data was compared with conventional sputum cytology for the diagnosis of lung cancer. All endogenous miRNAs were present in sputum in a remarkably stable form and sensitively and specifically detected by real-time RT-PCR. Mir-21 expression in the sputum specimens was significantly higher in cancer patients (76.32+/-9.79) than cancer-free individuals (62.24+/-3.82) (P<0.0001). Furthermore, overexpression of mir-21 showed highly discriminative receiver-operator characteristic (ROC) curve profile, clearly distinguishing cancer patients from cancer-free subjects with areas under the ROC curve at 0.902+/-0.054. Detection of mir-21 expression produced 69.66% sensitivity and 100.00% specificity in diagnosis of lung cancer, as compared with 47.82% sensitivity and 100.00% specificity by sputum cytology. The measurement of altered miRNA expression in sputum could be a useful noninvasive approach for the diagnosis of lung cancer., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

Background: Infection with the human immunodeficiency virus (HIV) and lung cancer represent two problems beginning in the 20th century that are of epidemic proportions. By the end of the 20th century, therapeutic programs of modest efficacy had been developed for both. Because both HIV infection and lung cancer are common, it is not surprising that a number of patients would be afflicted with both diseases simultaneously. There is a very limited literature regarding the treatment and outcome of patients with both diseases, particularly since the advent of highly active antiretroviral therapy (HAART) for HIV infection., Methods: We retrospectively reviewed our tumor registry to ascertain cases with concurrent lung cancer and HIV diagnoses since 1996, at the advent of HAART. Twenty-nine patients were identified at the University of Maryland, and five additional cases from an affiliated institution were identified., Results: Thirty patients had non-small cell lung cancer, and four patients had small cell lung cancer. Of the 30 patients with non-small cell lung cancer, 27 had stage IIIb/IV disease and were analyzed for outcome on the basis of CD4 counts and HAART therapy. Patients with CD4 counts >200 or those on HAART had numerically, though not statistically, superior survival. Patients were able to receive standard chemotherapy regimens, and the overall survival was 5.2 months., Conclusions: This single-institution analysis appears to indicate that there is an increasing incidence of patients with lung cancer and HIV infection. Patients with advanced NSCLC who are HIV positive with CD4 counts >200 can be treated with chemotherapy and demonstrate survival comparable to that of patients without HIV infection.