LncRNAs and EGFRvIII sequestered in TEPs enable blood-based NSCLC diagnosis

Abstract

Background: Tissue biopsy-based cancer diagnosis has limitations because of the fact that tumor tissues are in constant evolution and extremely heterogeneous. The current study was aimed to examine whether tumor-educated blood platelets (TEPs) might be a potential all-in-one source for blood-based cancer diagnostics to overcome the limitations of conventional cancer biopsy.

Methods: In the present study, we evaluated the expression pattern of MAGI2 antisense RNA 3 (MAGI2-AS3) and ZNFX1 antisense RNA 1 (ZFAS1) in both plasma and platelets of 101 non-small-cell lung cancer (NSCLC) patients. Receiver operating characteristic (ROC) curve was generated to evaluate their diagnostic potential. In addition, epidermal growth factor receptor (EGFR) mutations were detected in DNA and RNA samples of platelets for companion diagnostics.

Results: Our results showed that the levels of MAGI2-AS3 and ZFAS1 in both plasma and platelets of NSCLC patients were significantly downregulated than those in healthy controls. A positive correlation of long noncoding RNA expression was observed between platelets and plasma (r=0.738 for MAGI2-AS3, r=0.751 for ZFAS1, respectively). By ROC analysis, we found that molecular interrogation of MAGI2-AS3 and ZFAS1 in TEPs and plasma can offer valuable diagnostic performance for NSCLC patients (area under the ROC curve [AUC] _MAGI2-AS3 = 0.853/0.892, and AUC _ZFAS1 =0.780/0.744 for diagnosing adenocarcinoma and squamous cell carcinoma cases from controls, respectively). Clinicopathologic characteristic analysis further revealed that MAGI2-AS3 level significantly correlated with tumor-node-metastasis (TNM) stage (p=0.001 in TEPs, p=0.003 in plasma), lymph-node metastasis (p=0.016 in TEPs, p=0.023 in plasma), and distant metastasis (p=0.045 in TEPs, p=0.045 in plasma), while ZFAS1 level was only correlated with TNM stage (p=0.005 in TEPs, p=0.044 in plasma). Furthermore, EGFRvIII RNA existed in both TEPs and plasma, but EGFR intracellular mutations cannot be detected in DNA of TEPs isolated from NSCLC.

Conclusion: Our data suggested that TEP is a promising source for NSCLC diagnosis and companion diagnostics.

Keywords: EGFR mutations; NSCLC; lncRNA-MAGI2-AS3; lncRNA-ZFAS1; tumor-educated platelets.

Figure 1

MAGI2-AS2 and ZFAS1 levels were downregulated in platelets and plasma samples derived from NSCLC patients. Notes: (A) MAGI2-AS3 was downregulated in adenocarcinoma and SCC platelets than that in control platelets (p<0.001). (B) MAGI2-AS3 was downregulated in adenocarcinoma and SCC plasma than that in control plasma (p<0.001). (C) Platelets’ MAGI2-AS3 level was positively correlated to the plasma MAGI2-AS3 expression. The correlation coefficient was 0.738 (p<0.0001). (D) ZFAS1 was downregulated in adenocarcinoma and SCC platelets than that in control platelets (p<0.001). (E) ZFAS1 was downregulated in adenocarcinoma and SCC plasma than that in control plasma (p<0.001). (F) Platelets’ ZFAS1 level was positively correlated to the plasma ZFAS1 expression. The correlation coefficient was 0.751 (p<0.0001). The relative expression level was calculated using 2^−ΔCT method; error bars represent the median with interquartile range. All data were analyzed using nonparametric test; NS, no significance. ***p<0.001, ****p<0.0001. Abbreviations: AD, adenocarcinoma; MAGI2-AS3, MAGI2 antisense RNA 3; SCC, squamous cell carcinoma; ZFAS1, ZNFX1 antisense RNA 1.

Figure 2

Diagnostic value of MAGI2-AS3 and ZFAS1 in adenocarcinoma and SCC. Notes: (A) The ROC curve analysis for the diagnostic value of MAGI2-AS3 in platelets from adenocarcinoma (AUC=0.853, 95% CI=0.789–0.918, p<0.0001) and (AUC=0.866, 95% CI=0.802–0.929, p<0.0001) in plasma. (B) The ROC curve analysis for the diagnostic value of MAGI2-AS3 in platelets from SCC (AUC=0.892, 95% CI=0.819–0.965, p<0.0001) and (AUC=0.887, 95% CI=0.813–0.961, p<0.0001) in plasma. (C) The ROC curve analysis for the diagnostic value of ZFAS1 in platelets from adenocarcinoma (AUC=0.780, 95% CI=0.701–0.858, p<0.0001) and (AUC=0.806, 95% CI=0.731–0.881, p<0.0001) in plasma. (D) The ROC curve analysis for the diagnostic value of ZFAS1 in platelets from SCC (AUC=0.744, 95% CI=0.641–0.848, p<0.0001) and (AUC=0.770, 95% CI=0.663–0.878, p<0.0001) in plasma. (E) The ROC curve analysis for the diagnostic value of merged MAGI2-AS3 and ZFAS1 in platelets from adenocarcinoma (AUC=0.908, 95% CI=0.853–0.963, p<0.0001) and (AUC=0.890, 95% CI=0.834–0.946, p<0.0001) in plasma. (F) The ROC curve analysis for the diagnostic value of merged MAGI2-AS3 and ZFAS1 in platelets from SCC (AUC=0.919, 95% CI=0.848–0.990, p<0.0001) and (AUC=0.902, 95% CI=0.833–0.972, p<0.0001) in plasma. Abbreviations: AD, adenocarcinoma; AUC, area under the ROC curve; MAGI2-AS3, MAGI2 antisense RNA 3; ROC, receiver operating characteristic; SCC, squamous cell carcinoma; ZFAS1, ZNFX1 antisense RNA 1.

Figure 3

ARMS-PCR for EGFR intracellular mutations detection. Notes: (A), (B), (C) EGFR intracellular mutation cannot be detected in NO.6, NO.11, and NO.12 patient platelets. (D), (E), and (F) EGFR intracellular mutation can be detected in NO.6, NO.11, and NO.12 patient plasma. Abbreviations: ARMS, amplification-refractory mutation system; EGFR, epidermal growth factor receptor.

Figure 4

EGFRvIII expression in platelets and plasma derived from NSCLC patients. Notes: (A) EGFRvIII expression in 3 NSCLC platelets and plasma. (B) GAPDH expression as positive control in patients’ platelets and plasma, three controls’ results are shown here as an example. (C) No EGFRvIII expression in 50 healthy controls, and four individuals’ results are shown here as an example. (D) GAPDH expression as positive control in healthy individuals’ platelets and plasma. Four individuals’ results are shown here as an example. Abbreviations: EGFR, epidermal growth factor receptor; NSCLC, non-small-cell lung cancer.

Figure 5

A schematic figure summarizing potential role of platelets in NSCLC. Notes: Platelets communicate with tumor cells through uptaking of tumor-associated biomolecules and thus may serve as a potential diagnostic marker for NSCLC. Abbreviations: AD, adenocarcinoma; NSCLC, non-small-cell lung cancer; SCC, squamous cell carcinoma.