Abstract
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.
Highlights
Targeted therapy with tyrosine kinase inhibitors (TKIs) against the human epidermal growth factor receptor (EGFR) is currently the most common form of personalised treatment for non-small-cell lung carcinoma (NSCLC)
NSCLC pathological stages were defined according to the tumour node metastasis (TNM) International Staging System [30]
The association between miR-504 expression and mutant EGFR was confirmed in a validated cohort of 105 lung adenocarcinoma tissue samples from The Cancer Genome Atlas: a high level of miR-504 expression was associated with EGFR mutations
Summary
Targeted therapy with tyrosine kinase inhibitors (TKIs) against the human epidermal growth factor receptor (EGFR) is currently the most common form of personalised treatment for non-small-cell lung carcinoma (NSCLC). Deletions in exon 19 and the point mutation L858R are termed ‘classical’ activating mutations These mutations result in increased EGFR kinase activity, leading to enhanced downstream events. Another point mutation in codon 790 [threonine-790 to methionine (T790M)] of exon 20 of the EGFR gene was reported in approximately 50–60% of all patients who acquired resistance to EGFR TKI therapy. This mutation is probably acquired during treatment, because it is rarely detected de novo in tumours from untreated patients. Most patients develop some resistance to EGFR-TKIs after 9–13 months of treatment
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