Serum microRNA biomarkers for detection of non-small cell lung cancer.
ABSTRACT: Non small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality world-wide and the majority of cases are diagnosed at late stages of disease. There is currently no cost-effective screening test for NSCLC, and the development of such a test is a public health imperative. Recent studies have suggested that chest computed tomography screening of patients at high risk of lung cancer can increase survival from disease, however, the cost effectiveness of such screening has not been established. In this Phase I/II biomarker study we examined the feasibility of using serum miRNA as biomarkers of NSCLC using RT-qPCR to examine the expression of 180 miRNAs in sera from 30 treatment naive NSCLC patients and 20 healthy controls. Receiver operating characteristic curves (ROC) and area under the curve were used to identify differentially expressed miRNA pairs that could distinguish NSCLC from healthy controls. Selected miRNA candidates were further validated in sera from an additional 55 NSCLC patients and 75 healthy controls. Examination of miRNA expression levels in serum from a multi-institutional cohort of 50 subjects (30 NSCLC patients and 20 healthy controls) identified differentially expressed miRNAs. A combination of two differentially expressed miRNAs miR-15b and miR-27b, was able to discriminate NSCLC from healthy controls with sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 100% in the training set. Upon further testing on additional 130 subjects (55 NSCLC and 75 healthy controls), this miRNA pair predicted NSCLC with a specificity of 84% (95% CI 0.73-0.91), sensitivity of 100% (95% CI; 0.93-1.0), NPV of 100%, and PPV of 82%. These data provide evidence that serum miRNAs have the potential to be sensitive, cost-effective biomarkers for the early detection of NSCLC. Further testing in a Phase III biomarker study in is necessary for validation of these results.
Project description:The subtypes of NSCLC have unique characteristics of pathogenic mechanism and responses to targeted therapies. Thus, non-invasive markers for diagnosis of different subtypes of NSCLC at early stage are needed.Based on the results from the screening and validation process, 3 miRNAs (miR-532, miR-628-3p and miR-425-3p) were found to display significantly different expression levels in early-stage lung adenocarcinoma, as compared to those in healthy controls. ROC analysis showed that the miRNA-based biomarker could distinguish lung adenocarcinoma from healthy controls with high AUC (0.974), sensitivity (91.5%), and specificity (97.8%). Importantly, these three miRNAs could also distinguish lung adenocarcinoma from lung benigh diseases and other subtypes of lung cancer.Two hundreds and one early-stage lung adenocarcinoma cases and one hundreds seventy eight age- and sex-matched healthy controls were recruited to this study. We screened the differentially expressed plasma miRNAs using TaqMan Low Density Arrays (TLDA) followed by three-phase qRT-PCR validation. A risk score model was established to evaluate the diagnostic value of the plasma miRNA profiling system.Taken together, these findings suggest that the 3 miRNA-based biomarker might serve as a novel non-invasive approach for diagnosis of early-stage lung adenocarcinoma.
Project description:BACKGROUND:Among emerging circulating biomarkers, miRNA has the potential to detect lung cancer and follow the course of the disease. However, miRNA analysis deserves further standardization before implementation into clinical trials or practice. Here, we performed international ring experiments to explore (pre)-analytical factors relevant to the outcome of miRNA blood tests in the context of the EU network CANCER-ID. METHODS:Cell-free (cfmiRNA) and extracellular vesicle-derived miRNA (EVmiRNA) were extracted using the miRNeasy Serum/Plasma Advanced, and the ExoRNeasy Maxi kit, respectively, in a plasma cohort of 27 NSCLC patients and 20 healthy individuals. Extracted miRNA was investigated using small RNA sequencing and hybridization platforms. Validation of the identified miRNA candidates was performed using quantitative PCR. RESULTS:We demonstrate the highest read counts in healthy individuals and NSCLC patients using QIAseq. Moreover, QIAseq showed 15.9% and 162.9% more cfmiRNA and EVmiRNA miRNA counts, respectively, in NSCLC patients compared to healthy control samples. However, a systematic comparison of selected miRNAs revealed little agreement between high-throughput platforms, thus some miRNAs are detected with one technology, but not with the other. Adding to this, 35% (9 of 26) of selected miRNAs in the cfmiRNA and 42% (11 of 26) in the EVmiRNA fraction were differentially expressed by at least one qPCR platform; about half of the miRNAs (54%) were concordant for both platforms. CONCLUSIONS:Changing of (pre)-analytical methods of miRNA analysis has a significant impact on blood test results and is therefore a major confounding factor. In addition, to confirm miRNA biomarker candidates screening studies should be followed by targeted validation using an independent platform or technology.
Project description:<h4>Objectives</h4>In our previous studies we reported a panel of 24 miRNAs that allowed discrimination between blood of lung tumor patients independent of the histological subtype and blood of healthy controls with an accuracy of 95.4% [94.9%-95.9%]. Here, we now separately analyzed the miRNA expression in blood of non-small cell lung cancer (NSCLC), including squamous cell lung cancer and adenocarcinoma, and small cell lung cancer (SCLC) patients.<h4>Patients and methods</h4>In total, we examined the expression levels of 1,205 miRNAs in blood samples from 20 patients from each of the three histological groups and determined differentially expressed miRNAs between histological subtypes and metastatic and non-metastatic lung cancer. We further determined the overlap of miRNAs expressed in each subgroup with the 24-miRNA signature of lung tumor patients.<h4>Results</h4>Based on a raw p-value < 0.05, only 18 blood-borne miRNAs were differentially expressed between patients with adenocarcinoma and with squamous cell lung carcinoma, 11 miRNAs between adenocarcinoma and SCLC, and 2 between squamous cell lung carcinoma and SCLC. Likewise, the comparison based on a fold change of 1.5 did not reveal major differences of the blood-borne miRNA expression pattern between NSCLC and SCLC. In addition, we found a large overlap between the blood-borne miRNAs detected in the three histological subgroups and the previously described 24-miRNA signature that separates lung cancer patients form controls. We identified several miRNAs that allowed differentiating between metastatic and non-metastatic tumors both in blood of patients with adenocarcinoma and in blood of patients with SCLC.<h4>Conclusion</h4>There is a common miRNA expression pattern in blood of lung cancer patients that does not allow a reliable further subtyping into NSCLC or SCLC, or into adenocarcinoma and squamous cell lung cancer. The previously described 24-miRNA signature for lung cancer appears not primarily dependent on histological subtypes. However, metastatic adenocarcinoma and SCLC can be predicted with 75% accuracy.
Project description:BACKGROUND:Cachexia, highly prevalent in patients with non-small cell lung cancer (NSCLC), impairs quality of life and is associated with reduced tolerance and responsiveness to cancer therapy and decreased survival. MicroRNAs (miRNAs) are small non-coding RNAs that play a central role in post-transcriptional gene regulation. Changes in intramuscular levels of miRNAs have been implicated in muscle wasting conditions. Here, we aimed to identify miRNAs that are differentially expressed in skeletal muscle of cachectic lung cancer patients to increase our understanding of cachexia and to allow us to probe their potential as therapeutic targets. METHODS:A total of 754 unique miRNAs were profiled and analysed in vastus lateralis muscle biopsies of newly diagnosed treatment-naïve NSCLC patients with cachexia (n = 8) and age-matched and sex-matched healthy controls (n = 8). miRNA expression analysis was performed using a TaqMan MicroRNA Array. In silico network analysis was performed on all significant differentially expressed miRNAs. Differential expression of the top-ranked miRNAs was confirmed using reverse transcription-quantitative real-time PCR in an extended group (n = 48) consisting of NSCLC patients with (n = 15) and without cachexia (n = 11) and healthy controls (n = 22). Finally, these miRNAs were subjected to univariate and multivariate Cox proportional hazard analysis using overall survival and treatment-induced toxicity data obtained during the follow-up of this group of patients. RESULTS:We identified 28 significant differentially expressed miRNAs, of which five miRNAs were up-regulated and 23 were down-regulated. In silico miRNA-target prediction analysis showed 158 functional gene targets, and pathway analysis identified 22 pathways related to the degenerative or regenerative processes of muscle tissue. Subsequently, the expression of six top-ranked miRNAs was measured in muscle biopsies of the entire patient group. Five miRNAs were detectable with reverse transcription-quantitative real-time PCR analysis, and their altered expression (expressed as fold change, FC) was confirmed in muscle of cachectic NSCLC patients compared with healthy control subjects: miR-424-5p (FC = 4.5), miR-424-3p (FC = 12), miR-450a-5p (FC = 8.6), miR-144-5p (FC = 0.59), and miR-451a (FC = 0.57). In non-cachectic NSCLC patients, only miR-424-3p was significantly increased (FC = 5.6) compared with control. Although the statistical support was not sufficient to imply these miRNAs as individual predictors of overall survival or treatment-induced toxicity, when combined in multivariate analysis, miR-450-5p and miR-451a resulted in a significant stratification between short-term and long-term survival. CONCLUSIONS:We identified differentially expressed miRNAs putatively involved in lung cancer cachexia. These findings call for further studies to investigate the causality of these miRNAs in muscle atrophy and the mechanisms underlying their differential expression in lung cancer cachexia.
Project description:There is a need to develop a colorectal cancer (CRC) screening test that is noninvasive, cost effective, and sensitive enough to detect preneoplastic lesions. This case-control study examined the feasibility of using circulating extracellular microRNAs (miRNAs) to differentiate a spectrum of colorectal neoplasia of various severity and hence for early detection of colorectal neoplasia. Archived serum samples of 10 normal controls and 31 cases, including 10 with nonadvanced adenoma, 10 with advanced adenoma, and 11 with CRC, were profiled for circulating miRNAs using next-generation sequencing. Multiple linear regression, adjusting for age, gender, and smoking status, compared controls and the 3 case groups for levels of 175 miRNAs that met stringent criteria for miRNA sequencing analysis. Of the 175 miRNAs, 106 miRNAs were downregulated according to severity of neoplasia and showed a relative decrease in the expression from controls to nonadvanced adenoma to advanced adenoma to CRC (Ptrend < 0.05). Pairwise group comparisons showed that 39 and 80 miRNAs were differentially expressed in the advanced adenoma and CRC groups compared with the controls, respectively. Differences in miRNA levels between the nonadvanced adenoma group and controls were modest. Our study found that expression of many miRNAs in serum was inversely correlated with the severity of colorectal neoplasia, and differential miRNA profiles were apparent in preneoplastic cases with advanced lesions, suggesting circulating miRNAs could serve as potential biomarkers for CRC screening.
Project description:Lung cancer is a devastating disease with overall bleak prognosis. Current methods to diagnose lung cancer are rather invasive and are inadequate to detect the disease at an early stage when treatment is likely to be most effective. In this study, a shotgun sequencing approach was used to study the microRNA (miRNA) cargo of serum-derived exosomes of small cell lung cancer (SCLC) (n=9) and non-small cell lung cancer (NSCLC) (n=11) patients, and healthy controls (n=10). The study has identified 17 miRNA species that are differentially expressed in cancer patients and control subjects. Furthermore, within the patient groups, a set of miRNAs were differentially expressed in exosomal samples obtained before and after chemotherapy treatment. This manuscript demonstrates the potential of exosomal miRNAs for developing noninvasive tests for disease differentiation and treatment monitoring in lung cancer patients.
Project description:INTRODUCTION: MicroRNAs (miRNAs, miRs) are a class of small, non-coding RNA molecules with relevance as regulators of gene expression thereby affecting crucial processes in cancer development. MiRNAs offer great potential as biomarkers for cancer detection due to their remarkable stability in blood and their characteristic expression in many different diseases. We investigated whether microarray-based miRNA profiling on whole blood could discriminate between early stage breast cancer patients and healthy controls. METHODS: We performed microarray-based miRNA profiling on whole blood of 48 early stage breast cancer patients at diagnosis along with 57 healthy individuals as controls. This was followed by a real-time semi-quantitative Polymerase Chain Reaction (RT-qPCR) validation in a separate cohort of 24 early stage breast cancer patients from a breast cancer screening unit and 24 age matched controls using two differentially expressed miRNAs (miR-202, miR-718). RESULTS: Using the significance level of p<0.05, we found that 59 miRNAs were differentially expressed in whole blood of early stage breast cancer patients compared to healthy controls. 13 significantly up-regulated miRNAs and 46 significantly down-regulated miRNAs in our microarray panel of 1100 miRNAs and miRNA star sequences could be detected. A set of 240 miRNAs that was evaluated by radial basis function kernel support vector machines and 10-fold cross validation yielded a specificity of 78.8%, and a sensitivity of 92.5%, as well as an accuracy of 85.6%. Two miRNAs were validated by RT-qPCR in an independent cohort. The relative fold changes of the RT-qPCR validation were in line with the microarray data for both miRNAs, and statistically significant differences in miRNA-expression were found for miR-202. CONCLUSIONS: MiRNA profiling in whole blood has potential as a novel method for early stage breast cancer detection, but there are still challenges that need to be addressed to establish these new biomarkers in clinical use.
Project description:INTRODUCTION: Pulmonary arterial hypertension (PAH) is a major cause of mortality in systemic sclerosis (SSc). Screening guidelines for PAH recommend multiple investigations, including annual echocardiography, which together have low specificity and may not be cost-effective. We sought to evaluate the predictive accuracy of serum N-terminal pro-brain natriuretic peptide (NT-proBNP) in combination with pulmonary function tests (PFT) (‘proposed’ algorithm) in a screening algorithm for SSc-PAH. METHODS: We evaluated our proposed algorithm (PFT with NT-proBNP) on 49 consecutive SSc patients with suspected pulmonary hypertension undergoing right heart catherisation (RHC). The predictive accuracy of the proposed algorithm was compared with existing screening recommendations, and is presented as sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). RESULTS: Overall, 27 patients were found to have pulmonary hypertension (PH) at RHC, while 22 had no PH. The sensitivity, specificity, PPV and NPV of the proposed algorithm for PAH was 94.1%, 54.5%, 61.5% and 92.3%, respectively; current European Society of Cardiology (ESC)/European Respiratory Society (ERS) guidelines achieved a sensitivity, specificity, PPV and NPV of 94.1%, 31.8%, 51.6% and 87.5%, respectively. In an alternate case scenario analysis, estimating a PAH prevalence of 10%, the proposed algorithm achieved a sensitivity, specificity, PPV and NPV for PAH of 94.1%, 54.5%, 18.7% and 98.8%, respectively. CONCLUSIONS: The combination of NT-proBNP with PFT is a sensitive, yet simple and non-invasive, screening strategy for SSc-PAH. Patients with a positive screening result can be referred for echocardiography, and further confirmatory testing for PAH. In this way, it may be possible to shift the burden of routine screening away from echocardiography. The findings of this study should be confirmed in larger studies.
Project description:Nivolumab is an anti-PD1 antibody, given in second-line or later treatment in advanced non-small cell lung cancer (NSCLC). The objective of this study was to describe the predictive value of circulating tumor DNA (ctDNA) on the efficacy of nivolumab in advanced NSCLC. We prospectively included all consecutive patients with advanced NSCLC treated with nivolumab in our Department between June 2015 and October 2016. Plasma samples were obtained before the first injection of nivolumab and at the first tumor evaluation with nivolumab. ctDNA was analyzed by Next-Generation Sequencing (NGS), and the predominant somatic mutation was followed for each patient and correlated with tumor response, clinical benefit (administration of nivolumab for more than 6 months), and progression-free survival (PFS). Of 23 patients, 15 had evaluable NGS results at both times of analysis. ctDNA concentration at the first tumor evaluation and ctDNA change correlated with tumor response, clinical benefit and PFS. ROC curve analyses showed good diagnostic performances for tumor response and clinical benefit, both for ctDNA concentration at the first tumor evaluation (tumor response: positive predictive value (PPV) at 100.0% and negative predictive value (NPV) at 71.0%; clinical benefit: PPV at 83.3% and NPV 77.8%) and the ctDNA change (tumor response: PPV 100.0% and NPV 62.5%; clinical benefit: PPV 100.0% and NPV 80.0%). Patients without ctDNA concentration increase >9% at 2 months had a long-term benefit of nivolumab. In conclusion, NGS analysis of ctDNA allows the early detection of tumor response and long-term clinical benefit with nivolumab in NSCLC.
Project description:Selected circulating microRNAs (miRNAs) have been suggested for non-invasive screening of non-small cell lung cancer (NSCLC), however the numerous proposed miRNA signatures are inconsistent. Aiming to identify miRNAs suitable specifically for stage I-II NSCLC screening in serum/plasma samples, we searched the databases "Pubmed", "Medline", "Scopus", "Embase" and "WOS" and systematically reviewed the publications reporting quantitative data on the efficacy [sensitivity, specificity and/or area under the curve (AUC)] of circulating miRNAs as biomarkers of NSCLC stage I and/or II. The 20 studies fulfilling the search criteria included 1110 NSCLC patients and 1009 controls, and were of medium quality according to Quality Assessment of Diagnostic Accuracy Studies checklist. In these studies, the patient cohorts as well as the control groups were heterogeneous for demographics and clinicopathological characteristics; moreover, numerous pre-analytical and analytical variables likely influenced miRNA determinations, and potential bias of hemolysis was often underestimated. We identified four circulating miRNAs scarcely influenced by hemolysis, each featuring high sensitivity (> 80%) and AUC (> 0.80) as biomarkers of stage I-II NSCLC: miR-223, miR-20a, miR-448 and miR-145; four other miRNAs showed high specificity (> 90%): miR-628-3p, miR-29c, miR-210 and miR-1244. In a model of two-step screening for stage I-II NSCLC using first the above panel of serum miRNAs with high sensitivity and high AUC, and subsequently the panel with high specificity, the estimated overall sensitivity is 91.6% and overall specificity is 93.4%. These and other circulating miRNAs suggested for stage I-II NSCLC screening require validation in multiple independent studies before they can be proposed for clinical application.