The analysis of plasma cell-free DNA (cfDNA) is expected to provide useful biomarkers for early diagnosis of non-small-cell lung cancer (NSCLC). However, it remains unclear whether the intense release of cfDNA into the bloodstream of NSCLC patients results from malignancy or chronic inflammatory response. Consequently, the current diagnostic utility of plasma cfDNA quantification has not been thoroughly validated in subjects with chronic respiratory inflammation. Here we assess the effect of chronic respiratory inflammation on plasma cfDNA levels and evaluate the potential clinical value of this phenomenon as an early lung cancer diagnostic tool. We measured plasma cfDNA concentrations in 50 resectable NSCLC patients, 101 patients with chronic respiratory inflammation (chronic obstructive pulmonary disease, sarcoidosis, or asthma) and 40 healthy volunteers using real-time PCR. We found significantly higher plasma cfDNA levels in NSCLC patients than in subjects with chronic respiratory inflammation and healthy individuals (P2.8 ng ml(-1) provided 90% sensitivity and 80.5% specificity in discriminating NSCLC from healthy individuals (area under the curve (AUC)=0.90). The receiver-operating characteristics curve distinguishing NSCLC patients from subjects with chronic respiratory inflammation indicated 56% sensitivity and 91% specificity at the >5.25-ng ml(-1) cutoff (AUC=0.76). We demonstrated that elevated plasma cfDNA levels in NSCLC resulted primarily from tumour development rather than inflammatory response, raising the potential clinical implications for lung cancer screening and early diagnosis. Further research is necessary to better characterise and identify factors and processes regulating cfDNA levels in the blood under normal and pathological conditions.

Lung cancer is the most common cancer worldwide. Up to 85% of lung cancer cases are diagnosed as non-small cell lung cancer (NSCLC). The effectiveness of NSCLC treatment is expected to be improved through the implementation of robust and specific biomarkers. MicroRNAs (miRNAs) are small, non-coding molecules that play a key role in the regulation of basic cellular processes, including differentiation, proliferation and apoptosis, by controlling gene expression at the post-transcriptional level. Deregulation of miRNA activity results in the loss of homeostasis and the development of a number of pathologies, including lung cancer. During lung carcinogenesis, miRNAs exhibit dual regulatory function: they act as oncogenes to promote cancer development or as tumour suppressors. Unique miRNA sequences have been detected in malignant tissues and corresponding healthy tissues. Furthermore, stable forms of tumour-related miRNAs are detectable in the peripheral blood of patients with NSCLC. The potential benefits of using extracellular miRNAs present in body fluids as part of the diagnostic evaluation of cancer include low invasiveness (compared with tumour cell/tissue sampling), and the repeatability and ease of obtaining the specimens. Apart from the diagnostic applications of altered miRNA expression profiles, the dual regulatory role of miRNA in cancer might drive the further development of personalised therapies in NSCLC. The clinical usefulness of miRNA expression analysis to predict the efficacy of various treatment strategies including surgery, radio- and chemotherapy, and targeted therapies has been evaluated in NSCLC. Also, the capacity of a single miRNA to regulate the expression of multiple genes simultaneously presents an opportunity to use these small molecules in personalised therapy as individualised therapeutic tools. Open image in new window

Objective: Minute amounts of free-circulating DNA are present in plasma of healthy individuals, whereas its increased concentration was observed in patients with malignant tumors including non-small cell lung cancer (NSCLC). This study aimed at demonstrating the potential usefulness of plasma DNA concentration monitoring in NSCLC patients for therapy effectiveness assessment throughout the treatment and follow-up period. Methods: Plasma DNA concentration was assessed in 50 NSCLC patients (stage I – IIIA) prior and following the radical treatment using real-time quantitative PCR method. 10 orthopedic patient undergoing hip joint surgery and 40 healthy volunteers comprised control groups. Results: NSCLC patients (8.02 ng/ml) demonstrated significantly higher mean plasma DNA concentration with respect to healthy controls (2.27 ng/ml; p < 0.0000). Drastic increase in plasma DNA levels up to mean 68.74 ng/ml was detected a week after primary tumor resection. Still, similar phenomenon was observed in patients subjected to orthopedic surgical treatment (from 3.00 to 28.38 ng/ml, p < 0.0015). Most resected NSCLC patients with no disease recurrence during 3- to 6-month follow-up demonstrated reduced plasma DNA levels (mean 2.77 ng/ml) with respect to their presurgical values, whereas in relapsed subjects plasma DNA levels were significant higher. Conclusion: Free-circulating DNA concentration in plasma was significantly higher in NSCLC patients versus healthy controls. Its drastic increase following radical NSCLC treatment was most likely due to the surgical trauma. Importantly, the kinetics of plasma free-circulating DNA seems to be a promising marker of long-term effects of radical surgery in NSCLC.

MicroRNAs (miRNAs), key regulators of gene expression at the post-transcriptional level, are grossly misregulated in some human cancers, including non-small-cell lung carcinoma (NSCLC). The aberrant expression of specific miRNAs results in the abnormal regulation of key components of signalling pathways in tumour cells. MiRNA levels and the activity of the gene targets, including oncogenes and tumour suppressors, produce feedback that changes miRNA expression levels and indicates the cell’s genetic activity. In this study, we measured the expression of five circulating miRNAs (miR-195, miR-504, miR-122, miR-10b and miR-21) and evaluated their association with EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) mutation status in 66 NSCLC patients. Moreover, we examined the discriminative power of circulating miRNAs for EGFR mutant‐positive and -negative NSCLC patients using two different data normalisation approaches. We extracted total RNA from the plasma of 66 non-squamous NSCLC patients (31 of whom had tumours with EGFR mutations) and measured circulating miRNA levels using quantitative reverse transcription polymerase chain reaction (RT-qPCR). The miRNA expression levels were normalised using two endogenous controls: miR-191 and miR-16. We found significant associations between the expression of circulating miR-504 and EGFR-activating mutations in NSCLC patients regardless of the normalisation approach used (p = 0.0072 and 0.0236 for miR-16 and miR-191 normalisation, respectively). The greatest discriminative power of circulating miR-504 was observed in patients with EGFR exon 19 deletions versus wild-type EGFR normalised to miR-191 (area under the curve (AUC) = 0.81, p < 0.0001). Interestingly, circulating miR-504 levels were significantly reduced in the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutated subgroup compared to EGFR-mutated patients (p < 0.0030) and those with EGFR/KRAS wild-type tumours (p < 0.0359). Our study demonstrated the feasibility and potential diagnostic value of plasma miR-504 expression analysis to distinguish between EGFR-mutated and wild-type NSCLC patients. However, quality control and normalisation strategies are very important and have a major impact on the outcomes of circulating miRNA analyses.