MicroRNA Based Liquid Biopsy: The Experience of the Plasma miRNA Signature Classifier (MSC) for Lung Cancer Screening

Abstract

The development of a minimally invasive test, such as liquid biopsy, for early lung cancer detection in its preclinical phase is crucial to improve the outcome of this deadly disease. MicroRNAs (miRNAs) are tissue specific, small, non-coding RNAs regulating gene expression, which may act as extracellular messengers of biological signals derived from the cross-talk between the tumor and its surrounding microenvironment. They could thus represent ideal candidates for early detection of lung cancer. In this work, a methodological workflow for the prospective validation of a circulating miRNA test using custom made microfluidic cards and quantitative Real-Time PCR in plasma samples of volunteers enrolled in a lung cancer screening trial is proposed. In addition, since the release of hemolysis-related miRNAs and more general technical issues may affect the analysis, the quality control steps included in the standard operating procedures are also presented. The protocol is reproducible and gives reliable quantitative results; however, when using large clinical series, both pre-analytical and analytical features should be cautiously evaluated.

Circulating miRNAs and PD-L1 Tumor Expression Are Associated with Survival in Advanced NSCLC Patients Treated with Immunotherapy: a Prospective Study

“…The MSC test was performed following previously reported standard operating procedures with fixed parameters for prospective studies (25). Briefly, the effect of hemolysis on the MSC test was evaluated by both spectrophotometric and molecular analyses.…”

“…Samples with a 414 nm/375 nm absorbance ratio higher than 1.4 and in which at least 50% of the miRNA ratios exceeded the respective cut-off values were considered hemolyzed and excluded from further analysis. For all samples passing quality control, the four signatures of risk of disease (RD), presence of disease (PD), risk of aggressive disease (RAD), and presence of aggressive disease (PAD) were determined (25) (13).…”

“…The MSC was evaluable in 111 (79%) patients: 26 (19%) were MSC high risk, 51 (36%) were MSC intermediate, and 34 (24%) were MSC low risk. A total of 29 (20%) samples were not analyzable due to high hemolysis levels (25). Adequate tumor tissue samples for PD-L1 expression by 22C3 IHC assay were available for 104 (74%) patients: PNCs were <1% in 30 (21%) patients, between 1% and 49% in 36 (26%) patients, and !50% in 38 (27%) patients.…”

Plasma Next Generation Sequencing and Droplet Digital-qPCR-Based Quantification of Circulating Cell-Free RNA for Noninvasive Early Detection of Cancer

“…miRNAs have been also tested in several studies on a plethora of platforms, including microarray, next-generation sequencing (NGS), and RT-PCR. While NGS and microarray-based approaches are highly reproducible and specific, they lack the accuracy and sensitivity required for early detection, especially in the context of low copy transcripts [22].…”

Noncoding RNAs and Liquid Biopsy in Lung Cancer: A Literature Review

“…With NGS being still quite expensive and microarray a nonquantitative method with necessary experimental validation, RT-qPCR followed by TaqMan PCR is currently the most widely used assay for detection of circulating miRNA. The disadvantage of this technology is that, despite being accurate and sensitive, it can only detect already known miRNAs [118].…”