Combinatorial Blood Platelets-Derived circRNA and mRNA Signature for Early-Stage Lung Cancer Detection.

Despite the diversity of liquid biopsy transcriptomic repertoire, numerous studies often exploit only a single RNA type signature for diagnostic biomarker potential. This frequently results in insufficient sensitivity and specificity necessary to reach diagnostic utility. Combinatorial biomarker approaches may offer a more reliable diagnosis. Here, we investigated the synergistic contributions of circRNA and mRNA signatures derived from blood platelets as biomarkers for lung cancer detection. We developed a comprehensive bioinformatics pipeline permitting an analysis of platelet-circRNA and mRNA derived from non-cancer individuals and lung cancer patients. An optimal selected signature is then used to generate the predictive classification model using machine learning algorithm. Using an individual signature of 21 circRNA and 28 mRNA, the predictive models reached an area under the curve (AUC) of 0.88 and 0.81, respectively. Importantly, combinatorial analysis including both types of RNAs resulted in an 8-target signature (6 mRNA and 2 circRNA), enhancing the differentiation of lung cancer from controls (AUC of 0.92). Additionally, we identified five biomarkers potentially specific for early-stage detection of lung cancer. Our proof-of-concept study presents the first multi-analyte-based approach for the analysis of platelets-derived biomarkers, providing a potential combinatorial diagnostic signature for lung cancer detection.

Digital multiplexed analysis of circular RNAs in FFPE and fresh non-small cell lung cancer specimens.

Although many studies highlight the implication of circular RNAs (circRNAs) in carcinogenesis and tumor progression, their potential as cancer biomarkers has not yet been fully explored in the clinic due to the limitations of current quantification methods. Here, we report the use of the nCounter platform as a valid technology for the analysis of circRNA expression patterns in non-small cell lung cancer (NSCLC) specimens. Under this context, our custom-made circRNA panel was able to detect circRNA expression both in NSCLC cells and formalin-fixed paraffin-embedded (FFPE) tissues. CircFUT8 was overexpressed in NSCLC, contrasting with circEPB41L2, circBNC2, and circSOX13 downregulation even at the early stages of the disease. Machine learning (ML) approaches from different paradigms allowed discrimination of NSCLC from nontumor controls (NTCs) with an 8-circRNA signature. An additional 4-circRNA signature was able to classify early-stage NSCLC samples from NTC, reaching a maximum area under the ROC curve (AUC) of 0.981. Our results not only present two circRNA signatures with diagnosis potential but also introduce nCounter processing following ML as a feasible protocol for the study and development of circRNA signatures for NSCLC.

Multiplex Analysis of CircRNAs from Plasma Extracellular Vesicle-Enriched Samples for the Detection of Early-Stage Non-Small Cell Lung Cancer.

BACKGROUND: The analysis of liquid biopsies brings new opportunities in the precision oncology field. Under this context, extracellular vesicle circular RNAs (EV-circRNAs) have gained interest as biomarkers for lung cancer (LC) detection. However, standardized and robust protocols need to be developed to boost their potential in the clinical setting. Although nCounter has been used for the analysis of other liquid biopsy substrates and biomarkers, it has never been employed for EV-circRNA analysis of LC patients. METHODS: EVs were isolated from early-stage LC patients (n = 36) and controls (n = 30). Different volumes of plasma, together with different number of pre-amplification cycles, were tested to reach the best nCounter outcome. Differential expression analysis of circRNAs was performed, along with the testing of different machine learning (ML) methods for the development of a prognostic signature for LC. RESULTS: A combination of 500 µL of plasma input with 10 cycles of pre-amplification was selected for the rest of the study. Eight circRNAs were found upregulated in LC. Further ML analysis selected a 10-circRNA signature able to discriminate LC from controls with AUC ROC of 0.86. CONCLUSIONS: This study validates the use of the nCounter platform for multiplexed EV-circRNA expression studies in LC patient samples, allowing the development of prognostic signatures.