Non-small Cell Lung Cancer Serum Samples Research Articles

Serum transfer RNA-derived fragment tRF-31-79MP9P9NH57SD acts as a novel diagnostic biomarker for non-small cell lung cancer.

BackgroundtRNA‐derived fragments (tRFs) have been found to have a crucial function in the pathophysiology of cancers. However, the function of tRFs in non‐small cell lung cancer (NSCLC) is yet unknown. The goal of this study was to assess the tRF‐31‐79MP9P9NH57SD serum expression from NSCLC patients and to determine its diagnostic usefulness.MethodsBy using stem‐loop quantitative real‐time PCR, we were able to detect various tRF‐31‐79MP9P9NH57SD expressions in 96 NSCLC serum samples, 96 healthy controls, and 20 pairs of NSCLC serum samples pre‐ and post‐surgery (qRT‐PCR). After that, we analyzed its diagnostic effectiveness using the receiver operating characteristic (ROC) curve.ResultsSerum tRF‐31‐79MP9P9NH57SD expression was higher in NSCLC patients, and levels of tRF‐31‐79MP9P9NH57SD were linked to the clinical stage (p = 0.002) and the malignancy of lymph node (p = 0.012). In addition, after the procedure, the serum tRF‐31‐79MP9P9NH57SD expression in NSCLC patients dropped. With 48.96 percent sensitivity and 90.62 percent specificity, the area under ROC curve (AUC) was 0.733.Conclusionserum tRF‐31‐79MP9P9NH57SD possibly is a new and groundbreaking biomarker for the NSCLC.

Downregulation of long non-coding RNA LINC-PINT serves as a diagnostic and prognostic biomarker in patients with non-small cell lung cancer.

Long non-coding RNAs (lncRNAs) play an important role in gene regulation. Several lncRNAs have been demonstrated to be associated with the diagnosis and prognosis of non-small cell lung cancer (NSCLC). The present study aimed to investigate the role of lncRNA long intragenic non-protein-coding RNA p53-induced transcript (LINC-PINT) in NSCLC to identify a novel non-invasive biomarker for the diagnosis and prognosis of patients with NSCLC. Reverse transcription-quantitative PCR analysis was performed to detect LINC-PINT expression in the tissue and serum samples of patients with NSCLC. The diagnostic and prognostic values of LINC-PINT were assessed via the receiver operating characteristic curve, and Kaplan-Meier and Cox regression analyses, respectively. The results demonstrated that LINC-PINT expression was significantly downregulated in NSCLC serum samples and tissues. In addition, serum LINC-PINT exhibited diagnostic value in patients with NSCLC, and may be used to predict prognosis. Furthermore, aberrant LINC-PINT expression in tumor tissues was significantly associated with lymph node metastasis, tumor size, differentiation and TNM stage. Taken together, the results of the present study suggest that lncRNA LINC-PINT may be an independent diagnostic and prognostic biomarker in NSCLC.

Downregulation of oncogenic gene TGFβR2 by miRNA-107 suppresses non-small cell lung cancer

Non-small cell lung cancer (NSCLC) accounts for more than 80% of lung cancer cases with a low 5-year survival rate. MicroRNA may be targeted in the clinical treatment of the disease. In this study, miR-107 showed low expression in NSCLC serum samples, and it could suppress cell proliferation, migration and arrest cell cycle in NSCLC cell lines. Results revealed that miR-107 could inhibit the expression of transforming growth factor β receptor 2 (TGFβR2) via targeting TGFβR2. Downregulation of TGFβR2 also suppressed cell proliferation, migration and cell cycle in NSCLC cell lines. Our data suggested that miR-107 could inhibit the progression of NSCLC by targeting TGFβR2.

The expression of miR-424*in vivo and in vitro irradiated A549 cells, tissue and serum samples of non-small cell lung cancer

Objective To investigate the expression of miR-424* in 2 and 4 Gy X-ray irradiated A549 cells in vitro and in vivo, as well as in clinical lung tissues and serum sample of non-small cell lung cancer(NSCLC) patients, and to explore its potential role in the diagnosis and prognosis of lung cancer. Methods A549 cells were irradiated with 2 and 4 Gy X-rays, and some of irradiated cells were injected into nude mice through tail vein. Real time quantitative PCR (RT-qPCR) assay was employed to detect the expression of miR-424* in 2 and 4 Gy X-ray irradiated A549 cells in vitro and in vivo, as well as in clinical lung tissues and serum sample of lung cancer patients. Results Compared with the control group, the expression of miR-424* was up-regulated significantly in X-ray irradiated A549 cells at 1, 2, 12, 24 and 48 h post irradiation, respectively (2 Gy: t=-45.886--6.709, P<0.05; 4 Gy: t=-29.087--7.833, P<0.05). Furthermore, the expression of miR-424* was up-regulated in the lung and serum of nude mice with injection of 0, 2 and 4 Gy X-ray irradiated A549 cells, compared with control group (fold change was 9.72, 8.58 and 4.7 with 2 Gy irradiation and 11.93, 9.22 and 8.99 with 4 Gy irradiation, t=-13.243, -12.409, -9.833 in lung and t=-6.436, -3.052, -3.609 in serum, respectively, P<0.05). Out of 11 tissue samples of NSCLC patients, 6 were detected with up-regulated miR-424* expression, and no significant discrepancy of miR-424* expression was detected in two type of NSCLC tissue samples. On the contrary, 43 serum samples were detected with up-regulated miR-424* expression out of 84 serum samples (51.20%) of NSCLC patients (fold change range 1.97 to 17.71), and significant discrepancy of miR-424* expression was shown in two subtypes of NSCLC serum samples [adenocarcinoma: 39.10% (18/46) and squamous carcinoma: 65.8% (25/38)], as well as in serum samples of NSCLC patients with radiotherapy [41.5% (22/53)] and without radiotherapy [67.7% (21/31)] (t=5.919, 5.387, P<0.05, respectively). Conclusions 2 and 4 Gy X-ray irradiation could up-regulate the expression of miR-424* in A549 cells, which might be correlated with the enhanced metastasis of A549 cells induced by X-ray in vivo and in vitro. Furthermore, the expression of miR-424* was up-regulated in over 50% of the tissue and serum samples of NSCLC patients, which might be correlated with the diagnosis of NSCLC subtype and prognosis of radiotherapy. Key words: Non-small cell lung cancer (NSCLC); miR-424*; Irradiation; microRNA

P2.03b-024 microRNA-330-3p Promotes Brain Metastasis of Non-Small Cell Lung Cancer (NSCLC) by Activating MAPK/MEK/ERK Signaling Pathway: Topic: Biomarkers

Brain metastasis (BM) is associated with poor prognosis, recurrence, and death in patients with non-small cell lung cancer (NSCLC). MicroRNAs (miRNAs) play an essential role in the development of NSCLC. We investigated miRNAs that serve as biomarkers to differentiate NSCLC patients with and without BM, and explored the underlying mechanism. The significant association for BM of NSCLC was analyzed by univariate and multivariable logistic regression analysis of the clinical features of NSCLC patients with BM and without BM. The effects of miR-330-3p on NSCLC cells (A549 and HCC827) were investigated using assays of cell viability, migration, invasion, cell cycle, Western blot, and a tumor xenograft and brain metastatic xenografts models. The miR-330-3p targets were identified using microarray analysis and verified by luciferase reporter assay. Female gender (OR = 3.15，P = .003), age under 60 (OR = 4.54, P < .001), adenocarcinoma (OR = 3.57, P = .01), EGFR19 exon mutation (OR = 3.76, P = .05), N2 (OR = 8.23, P = .01) or N3 (OR = 29.38, P < .001) were highly associated with BM in NSCLC patients. The miR-330-3p was expressed at a higher level in BM+ than in BM- NSCLC serum samples, and also higher in NSCLC cell lines than the normal human bronchial epithelial cell line. Cell proliferation, migration and invasiveness of 2 representative NSCLC cell lines (A549 and HCC827) were increased by over-expressing miR-330-3p using a lentivirus carrying a sequence of miR-330-3p, and decreased by knockdown of miR-330-3p. In nude mice receiving subcutaneous A549 and HCC827 cell inoculation, tumor growth were significantly faster in mice receiving A549 and HCC827 cell permanently expressing exogenous miR-330-3p, and slower in cells permanently expressing an anti- miR-330-3p sequence. The mice receiving cancer cells stably expressing exogenous miR-330-3p injection directly into the brain almost developed multiple metastatic foci, while developed a smaller orthotopic tumor in mice receiving injection of cells expressing an anti- miR-330-3p sequence. GRIA3 was identified as a direct target of miR-330-3p using luciferase reporter assays. Real-time PCR and Western blot confirmed that miR-330-3p downregulated GRIA3 expression. MEK inhibition suggested that GRIA3 was regulated by miR-330-3p via MAPK/MEK/ERK signaling pathway. These results support the oncogenic role of miR-330-3p in NSCLC brain metastasis, providing a rationale for miRNA-targeted therapeutic strategies.

Abstract 5027: Detection of soluble protein biomarkers in NSCLC serum samples by meso scale discovery electrochemiluminescence platform

Abstract Identification of soluble biomarkers has become a critical non-invasive approach for disease diagnosis and monitoring in the treatment of solid tumors. Non-Small Cell Lung Cancer (NSCLC) is the most common form of lung cancer, accounting for approximately 85% of all lung cancers worldwide. The EGFR family of genes is known to be highly expressed in NSCLC tumors and play a critical role in the progression of the disease. Although patients initially respond well to EGFR TKI therapy, acquired resistance occurs through several mechanisms, including compensatory cMET pathway activation (Robinson et al. 2013). Previous reports have demonstrated that soluble forms of EGFR, Her2 and cMET are generated through either alternate splicing of mRNA or proteolytic cleavage of the full length receptors (Wilken et al. 2013). Serum concentrations of these receptors can be correlated with prognosis as well as treatment response (Gregorc, 2004, Kasahara, 2010 Heinmoller, 2003). Soluble receptor ligands, including Hepatocyte growth factor (HGF), have also been evaluated in NSCLC and a strong correlation has also been observed between the levels of serum HGF and disease outcome in patients treated with EGFR-TKIs (Kasahara et al. 2010). In this study, we evaluated concentrations of sEGFR, sMET, sHER2,, and HGF in NSCLC versus healthy normal serum samples via meso scale discovery (MSD) platform. Inventoried and custom MSD assays were optimized to measure these proteins in a training set consisting of 19 NSCLC and 16 normal healthy serum samples. Significantly lower concentrations of sEGFR and sHER2 were observed in the NSCLC serum samples compared to normal (p value = 0.0092 for both receptors), whereas, in contrast, the levels of HGF were significantly higher in the NSCLC patient samples (p value &amp;lt;0.0001). No significant difference in sMET concentration was observed between NSCLC and normal serum. Based on this data, it was determined that a ratio of protein concentration of each soluble receptor (EGFR, Her2 and cMET) to HGF yielded a greater differentiation between NSCLC and normal samples (p value &amp;lt;0.0001, = 0.0041,and 0.0327 respectively). Cut-off values to define NSCLC versus normal patients were established for each ratio and validated in a blinded independent cohort, consisting of 50 NSCLC and 12 healthy normal serum samples. PPV and NPV were then calculated to determine sensitivity and specificity for each protein ratio. Results from the validation study show an almost complete separation between healthy and NSCLC patient samples (p value &amp;lt;0.0001, AUC = 1)for all three ratios tested (sEGFR/HGF, sHER2/HGF and sMET/HGF), with a PPV = 100%, 98%, 100% and NPV = 100%, 100%, 85.7%, respectively. Overall, these findings indicate potential clinical applications for these protein pairs as non-invasive biomarkers of NSCLC diagnosis, prognosis and treatment monitoring to support emerging TKI therapies Citation Format: Dana S. Gaffney, Katherine Bell, Gabriela Martinez, Jayaprakash Karkera, Suso Platero. Detection of soluble protein biomarkers in NSCLC serum samples by meso scale discovery electrochemiluminescence platform. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5027.

Serum microRNA-365 in combination with its target gene TTF-1 as a non-invasive prognostic marker for non-small cell lung cancer.

MicroRNA (miR)-365 functions as a tumor suppressor in non-small cell lung cancer (NSCLC) cells by targeting thyroid transcription factor 1 (TTF-1). To investigate miR-365 and TTF-1 mRNA expression in serum of NSCLC and their associations with patients' prognosis. MiR-365 and TTF-1 mRNA expression in 100 NSCLCs and 100 healthy control sera were detected by quantitative real-time PCR (qRT-PCR). MiR-365 expression level was significantly lower in NSCLC serum samples than in healthy control serum samples (P<0.001), while TTF-1 mRNA expression level was significantly increased in NSCLC serum samples compared to healthy control serum samples (P<0.001). In addition, low miR-365 expression and high TTF-1 expression, alone or in combination, were all significantly associated with poor differentiation (P=0.008, 0.008 and 0.001, respectively), advanced TNM stage (P=0.001, 0.005 and <0.001 respectively) and positive lymph node metastasis (P=0.02, 0.02 and 0.01, respectively) of NSCLC patients. Notably, NSCLC patients with combined low miR-365 expression and high TTF-1 expression (miR-365-low/TTF-1-high) had shortest overall survival (P<0.001). Furthermore, multivariate analysis showed that miR-365 expression (P=0.01), TTF-1 expression (P=0.01), and combined expression of miR-365 and TTF-1 (miR-365/TTF-1, P=0.001) were all independent prognostic factors for overall survival in NSCLC patients. Our data reveal that preoperative serum miR-365 and TTF-1 mRNA levels may be both effective indicators of tumor aggressiveness in human NSCLC. More interestingly, miR-365 and its target gene TTF-1 appear to be synergistic risk factors for the reduction in overall survival of patients with NSCLC.

Prognostic potential of microRNA-138 and its target mRNA PDK1 in sera for patients with non-small cell lung cancer.

microRNA (miR)-138 has been recognized as a potential tumor suppressor via regulating 3-phosphoinositide-dependent protein kinase-1 (PDK1) expression in non-small cell lung cancer (NSCLC) cells. The aim of this study was to investigate miR-138 and PDK1 mRNA expression in serum of NSCLC and their associations with patients' prognosis. miR-138 and PDK1 mRNA expressions in 100 NSCLCs and 100 healthy control sera were detected by quantitative real-time PCR. miR-138 expression level was significantly lower in NSCLC serum samples than in healthy control serum samples (P < 0.001), while PDK1 mRNA expression level was significantly increased in NSCLC serum samples compared to healthy control serum samples (P < 0.001). In addition, miR-138 downregulation and PDK1 upregulation were both significantly associated with advanced tumor-node-metastasis (TNM) stage (both P = 0.002) and positive lymph node metastasis (both P = 0.01) of NSCLC patients. Moreover, the overall survival of NSCLC patients with low miR-138 expression or high PDK1 mRNA expression was obviously shorter than those with high miR-138 expression or low PDK1 mRNA expression (both P < 0.001). Notably, NSCLC patients with combined miR-138 downregulation and PDK1 upregulation (miR-138-low/PDK1-high) had shortest overall survival (P < 0.001). Furthermore, multivariate analysis showed that miR-138 expression (P = 0.01), PDK1 expression (P = 0.01), and combined expression of miR-138 and PDK1 (miR-138/PDK1, P = 0.001) were all independent prognostic factors for overall survival in NSCLC patients. Deregulation of miR-138/PDK1 cascade may be implicated in carcinogenesis and cancer progression of human NSCLC. More importantly, miR-138 and PDK1 may synergistically predict patients' prognosis and their combination may represent a promising prognostic biomarker of human NSCLC.

Proteomics Characterization of Haptoglobin Alpha-2 Subunit in Non-Small Cell Lung Cancer Serum and Various Human Materials

Non-small cell lung cancer (NSCLC) is detected in all lung cancer patients and the serum samples as potential source are often used as a potential source for early diagnosis. In this study, we used the proteomic approaches combining 2-DE, 2-D DIGE and mass spectrometry to identify the potential serum biomarkers in NSCLC serum samples, and compare the result to normal serum samples. The haptoglobin alpha 2 subunit (HAP2) is one of the proteins we are interested in and showed the overexpression level in NSCLC serum amongst the NSCLC subtypes. The identity of HAP2 was confirmed by LC-MS/MS and NH2-terminal amino acid sequencing analyses, while the high expression level of HAP2 in NSCLC serum samples was validated using co-immunoprecipitation and western blotting. Interestingly, the high expression of HAP2 has specificity in human serum samples, but nonspecific expression in lung cancer tissues and is not observed in lung cancer cell lines except HepG2 hepatocellular carcinoma cell line. This indicates that HAP2 is not produced from lung tissues and/or cells, but may be secreted from the liver. In addition, HAP2 isoforms and its post-translational modifications (PTMs) could be detected by various staining methods such as phosphoprotein, glycoprotein stains and FITC-labeled lectins stain. The different carbohydrate specificities of HAP2 isoforms in NSCLC serum samples, especially sialyl Lewis x, was observed and may correlate to the development or metastasis of lung cancer. We suggest that HAP2 may become a potentially useful serum biomarker for early diagnostic and therapeutic applications.

Identification of ten serum microRNAs from a genome‐wide serum microRNA expression profile as novel noninvasive biomarkers for nonsmall cell lung cancer diagnosis

The detection of nonsmall cell lung cancer (NSCLC) at an early stage presents a daunting challenge due to the lack of a specific noninvasive marker. The discovery of microRNAs (miRNAs), particularly those found in serum, has opened a new avenue for tumor diagnosis. To determine whether the expression profile of serum miRNAs can serve as a NSCLC fingerprint, we performed Taqman probe-based quantitative RT-PCR assay to selected differentially expressed serum miRNAs from a sample set including 400 NSCLC cases and 220 controls, and risk score analysis to evaluate the diagnostic value of the serum miRNA profiling system. After a two-phase selection and validation process, 10 miRNAs were found to have significantly different expression levels in NSCLC serum samples compared with the control serum samples. Risk score analysis showed that this panel of miRNAs was able to distinguish NSCLC cases from controls with high sensitivity and specificity. Under ROC curves, the AUC for tumor identification in training set and validation set were 0.966 and 0.972, respectively. Furthermore, the expression profile of the 10-serum miRNAs was correlated with the stage of NSCLC patients, especially in younger patients and patients with current smoking habits. More importantly, the serum miRNA-based biomarker for early NSCLC detection was supported by a retrospective analysis in which the 10-serum miRNA profile could accurately classify serum samples collected up to 33 months ahead of the clinical NSCLC diagnosis. Taken together, we demonstrate that the profiling of 10-serum miRNAs provides a novel noninvasive biomarker for NSCLC diagnosis.

Prognostic significance of serum miRNA‐21 expression in human non‐small cell lung cancer

Deregulation of microRNAs (miRNAs) plays important roles in tumor progression. The aim of this study was to investigate miR-21 expression in serum of non-small cell lung cancer (NSCLC) and its correlation with prognosis of NSCLC patients. Dysregulated miRNAs in NSCLC serum were identified by microarray. MiR-21 expression in NSCLC and control serum was detected by TaqMan RT-PCR assay. The correlation of serum miR-21 with clinicopathological factors of NSCLC patients was analyzed. Furthermore, the prognostic significance of serum miR-21 was analyzed by using Kaplan-Meier curves with log-rank tests and a Cox proportional hazard model. The level of miR-21 expression was higher in NSCLC serum samples than in control serum samples (P < 0.01). High serum miR-21 was significantly correlated with tumor-node metastases stage and lymph node metastasis of NSCLC patients (P = 0.016 and 0.026, respectively). The 3-year actuarial overall survival rates in NSCLC patients with high serum miR-21 expression (39.8%) was significantly shorter than those with low serum miR-21 expression (58.2%; P < 0.001). Furthermore, univariate and multivariate analyses for overall survival showed that serum miR-21 expression was an independent prognostic factor for NSCLC patients (P = 0.015, RR = 2.01, 95% CI: 1.78-3.26). Serum miR-21 expression might be useful as a prognostic marker for NSCLC patients.

Examination of 12-lipoxygenase (12-LOX) as a therapeutic target in non-small cell lung cancer (NSCLC): Mechanisms controlling survival and induction of apoptosis following selective inhibition.

7068 Background: Platelet-type 12-LOX is an arachidonic acid metabolising enzyme resulting in the formation of 12(S)-HETE, which stimulates tumour cell adhesion, invasion and metastasis. This study aimed to examine the expression profile and role of this enzyme in NSCLC, and determine if it is a potential target for intervention. Methods: A panel of retrospective resected lung tumours was stained for 12-LOX expression by IHC. Levels of the 12-LOX metabolite, 12(S)-HETE, were examined in 50 NSCLC serum samples, and correlated with serum VEGF. A panel of NSCLC cell lines were treated with baicalein (10 uM), a selective inhibitor of 12-LOX, or 12(S)-HETE (100 ng/ml) and cell survival/proliferation examined by BrdU. Apoptosis following 12-LOX inhibition was examined by HCS and validated by FACS and DNA laddering. The effect of 12-LOX inhibition on NSCLC tumour growth and survival was examined in-vivo using an athymic nude mouse model. Gene alterations following 12-LOX inhibition in NSCLC cell lines were assessed by qPCR arrays and validated by RT-PCR. Transient transfection methods were used to examine the effects of 12-LOX overexpression in NSCLC cells. Results: 12-LOX expression was observed to a varying degree in human lung cancers of varying histological subtypes. 12(S)-HETE levels were correlated (p<0.05) with those of VEGF. Baicalein inhibited proliferation/survival in all cell lines, while 12(S)-HETE increased proliferation. 12-LOX inhibition increased apoptosis, indicated by a reduction in f-actin content and mitochondrial mass potential. Treatment with baicalein significantly reduced the growth of NSCLC tumours and increased overall survival in athymic nude mice. qPCR array data implicated a number of apoptosis/angiogenesis genes regulating these effects, including bcl-2, VEGF, integrin A2 and A4. 12-LOX overexpression resulted in an increase in VEGF secretion, confirming qPCR observations. Conclusions: 12-LOX is a survival factor/potential target in NSCLC, and correlated with angiogenic factor expression. Selective 12-LOX targeting may have clinical benefit for the future treatment.

Performance characteristics of an automated assay for the quantitation of CYFRA 21-1 in human serum

Objectives A fragment of cytokeratin 19, CYFRA 21-1, has been reported to be a sensitive tumor marker for non-small cell lung cancer (NSCLC). We describe analytical performance characteristics of a novel CYFRA 21-1 assay and hypothesize that CYFRA 21-1 complements the clinical sensitivity of carcinoembryonic antigen (CEA) and squamous cell carcinoma antigen (SCCa). Design and methods Performance characteristics of a CYFRA 21-1 immunochemiluminescent assay included analytical sensitivity, imprecision, linearity, analyte stability, and reference interval determination. Ninety-two pretreatment NSCLC serum samples were tested for CYFRA 21-1, CEA, and SCCa. Sensitivity was determined for each marker individually and in combination, with regard to tumor stage and histology. Results The analytical sensitivity was 0.01 ng/mL. Total imprecision ranged from 4.0 to 6.3% at 4.9 to 28.4 ng/mL, respectively. The assay was linear from 0.9 to 71.4 ng/mL (slope = 0.995, intercept = − 0.60, r 2 = 0.999). CYFRA 21-1 was stable for 48 h at ambient temperature and 14 days at 4 °C. The 97.5th percentile of a reference population was 1.9 ng/mL. Across disease stage, the sensitivities of CYFRA 21-1, CEA, and SCCa were 17–81%, 30–52%, and 24–39%, respectively. CYFRA 21-1 combined with CEA or SCCa increased sensitivity above that of any single marker. Conclusions An immunochemiluminescent assay for CYFRA 21-1 had favorable performance characteristics. CYFRA 21-1 was complementary to CEA and SCCa and increased clinical sensitivity in patients with NSCLC.

Abstract 3015: The miR-21:miR-221 ratio as a serum-based diagnostic for non-small cell lung cancer

Abstract MicroRNAs (miRNA) are small (21-25 nucleotides) non-coding regulatory RNAs that control protein expression at the transcriptional level through various mechanisms. Previous studies have shown that circulating miRNA can be utilized as a tool to gain a better understanding of both benign and malignant tumor conditions. Their altered expression has been linked with several cancers including lung cancer. Lung cancer is the leading cause of death worldwide with approximately 85% of diagnoses being attributed to non-small cell lung cancer (NSCLC). The goal of this study was to determine if the presence of circulating miRNAs from NSCLC patients can serve as non-invasive diagnostic biomarkers of disease and therapy monitoring. We initially evaluated the expression of 16 NSCLC-specific miRNAs (miR-146b, miR-155, miR-205, miR-21, miR-221, miR-199a-5p, miR-222, miR-320, miR-223, miR-25, miR-16, miR-191, miR-20a, miR-24, miR-145, and miR-152) in 46 NSCLC serum samples ranging from stage I to IV, including 20 adenocarcinoma and 26 squamous cell carcinoma samples. Eighteen healthy controls were used for comparison. RNA was extracted from serum using the single-step Trizol method and real-time PCR (Taqman). Of the 16 miRNAs tested, six miRNAs were significantly upregulated in the NSCLC when compared to the normals (p&amp;lt;0.0001 with AUROCs of 0.80 to 0.93). Top Scoring Pair analyses identified the ratio of miR-21 to miR-221 as the most robust predictor of NSCLC status compared to healthy subjects with an AUROC of 0.95 (95% CI; 0.91-0.99, p &amp;lt; 0.001) and 17% error rate (leave-one-out cross validation). This miRNA pair was then validated in an independent set of 19 NSCLC and 26 normal serum samples with overall accuracy of 80% (89% and 73% for NSCLC and normal, respectively) and AUC of 0.88 (95%CI: 0.78-0.98). These findings demonstrate the potential of using miR-21 and miR-221 in detecting NSCLC from the serum. Furthermore, these miRNAs could lead to the development of non-invasive biomarkers for early diagnosis, prognosis, and therapy monitoring in NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3015.