Development and Validation of Tumor-educated Blood Platelets Integrin Alpha 2b (ITGA2B) RNA for Diagnosis and Prognosis of Non-small-cell Lung Cancer through RNA-seq

Shan Xing, Tao Zeng, Ning Xue, Yi He, Yan-Zhen Lai, Hui-Lan Li, Qi Huang, Shu-Lin Chen, Wan-Li Liu, Shan Xing, Tao Zeng, Ning Xue, Yi He, Yan-Zhen Lai, Hui-Lan Li, Qi Huang, Shu-Lin Chen, Wan-Li Liu

Abstract

Background: Currently, there are no molecular biomarkers for the early detection of non-small-cell lung cancer (NSCLC). This study focused on identifying RNAs found on tumor-educated blood platelets (TEPs) for detecting stage I NSCLC. Methods: Platelet RNAs, isolated from the blood of 9 patients with NSCLC (stages I and II) and 8 healthy controls, were analyzed using RNA-seq. ITGA2B was selected as a candidate marker. Two different Polymerase Chain Reactions (PCR) were used to measure ITGA2B in platelet samples from healthy controls (n = 150), patients with NSCLC (n = 243), and patients with benign pulmonary nodules (n = 141) in two cohorts. Results: Platelet ITGA2B levels were significantly higher (p < 0.001) in patients with NSCLC than in all controls. The diagnostic accuracy of ITGA2B was area under the curve (AUC) of 0.922 [95% confidence interval (CI), 0.892-0.952], sensitivity of 92.8%, and specificity of 78.6% in the test cohort and 0.888, 91.2%, and 56.5% in the validation cohort for NSCLC by quantitative real time PCR (q-PCR). Furthermore, ITGA2B maintained diagnostic accuracy for patients with NSCLC using Droplet Digital PCR (ddPCR) and the other type of internal control, Ribosomal Protein L32 (RPL32) [ddPCR: 0.967 (0.929-1.000) and RPL32: 0.847(0.773-0.920)]. A nomogram incorporating ITGA2B, carcinoembryonic antigen (CEA) and stage could predict the overall survival (C-index = 0.756). Conclusions: TEP ITGA2B is a promising marker to improve identification of patients with stage I NSCLC and differentiate malignant from benign lung nodules.

Keywords: ITGA2B; NSCLC; diagnosis; survival; tumor-educated blood platelets.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Study profiles. NSCLC: non-small cell lung cancer; BPN: benign pulmonary nodules; HC: healthy controls; K-M analyses: Kaplan-Meier analyses; q-PCR: quantitative real-time PCR; ddPCR: Droplet digital PCR
Figure 2
Figure 2
Candidate platelet mRNA selection from RNA-seq analysis. (A) Volcano plot of mRNAs expression in 9 early stage NSCLC versus 8 HC. (B) Top 20 enrichment of GOs for differentially expressed mRNAs in NSCLC. Red for NSCLC, and blue for HC. (C) Bubble plot of top 10 enrichment of Pathways for differentially expressed mRNAs in NSCLC. (D) Venn diagram analysis of differentially expressed mRNAs from our dataset, GSE68086 and GSE89843. (E) mRNA-GO-pathway correlation network analysis of core mRNAs and their functions. NSCLC: non-small cell lung cancer; HC: healthy controls
Figure 3
Figure 3
Levels of platelet ITGA2B, SELP mRNA and serum CEA concentrations in the test and validation cohorts. The representative amplification curve of Real-time PCR reaction for ITGA2B (A) and SELP (B). Levels of ITGA2B for test cohort (C). ITGA2B for validation cohort (D). SELP for test cohort (E). SELP for validation cohort (F). CEA for test cohort (G). CEA for validation cohort (H). NSCLC: non-small cell lung cancer; BPN: benign pulmonary nodules; HC: healthy controls; ITGA2B: Integrin, Alpha 2b; SELP: P-selectin; CEA: carcinoembryonic antigen.
Figure 4
Figure 4
Diagnostic outcomes for platelet ITGA2B, SELP mRNA and serum CEA in the NSCLC. (A) ROC curve for platelet ITGA2B, SELP mRNA, serum CEA or combination of ITGA2B and CEA with NSCLC versus all controls in the test cohort. (B) ROC curve for platelet ITGA2B, SELP mRNA, serum CEA or combination of ITGA2B and CEA with NSCLC versus all controls in the validation cohort. (C) The rate of positive results for CEA, ITGA2B, or both, in all patients with NSCLC, and for ITGA2B by CEA status in the test cohort. (D) The rate of positive results for CEA, ITGA2B, or both, in all patients with NSCLC, and for ITGA2B by CEA status in the validation cohort. (E) ROC curve for platelet ITGA2B, SELP mRNA, serum CEA or combination of ITGA2B and CEA with NSCLC versus BPN in the test cohort. (F) ROC curve for platelet ITGA2B, SELP mRNA, serum CEA or combination of ITGA2B and CEA with NSCLC versus BPN in the validation cohort. (G) The rate of positive results for CEA and ITGA2B for patients with BPN, and for ITGA2B by CEA status in the test cohort. (H) The rate of positive results for CEA and ITGA2B for patients with BPN, and for ITGA2B by CEA status in the validation cohort. ROC: receiver operating characteristics; NSCLC: non-small cell lung cancer; BPN: benign pulmonary nodules; HC: healthy controls; C: CEA, carcinoembryonic antigen; I: ITGA2B, Integrin, Alpha 2b; SELP: P-selectin.
Figure 5
Figure 5
Diagnostic outcomes for platelet ITGA2B, SELP mRNA and serum CEA in the stage I NSCLC. (A) ROC curve for platelet ITGA2B, SELP mRNA, serum CEA or combination of ITGA2B and CEA with stage I NSCLC versus all controls in the test cohort. (B) ROC curve for platelet ITGA2B, SELP mRNA, serum CEA or combination of ITGA2B and CEA with stage I NSCLC versus all controls in the validation cohort. (C) The rate of positive results for CEA, ITGA2B, or both, in all patients with stage I NSCLC, and for ITGA2B by CEA status in the test cohort. (D) The rate of positive results for CEA, ITGA2B, or both, in all patients with stage I NSCLC, and for ITGA2B by CEA status in the validation cohort. (E) ROC curve for platelet ITGA2B, SELP mRNA, serum CEA or combination of ITGA2B and CEA with stage I NSCLC versus BPN in the test cohort. (F) ROC curve for platelet ITGA2B, SELP mRNA, serum CEA or combination of ITGA2B and CEA with stage I NSCLC versus BPN in the validation cohort. (G) ROC curve for platelet ITGA2B, SELP mRNA, serum CEA or combination of ITGA2B and CEA with stage I NSCLC with a solitary small tumour (≤ 2cm) versus BPN in the test cohort. (H) ROC curve for platelet ITGA2B, SELP mRNA, serum CEA or combination of ITGA2B and CEA with stage I NSCLC with a solitary small tumour (≤ 2cm) versus BPN in the validation cohort. ROC: receiver operating characteristics; NSCLC: non-small cell lung cancer; BPN: benign pulmonary nodules; HC: healthy controls; C: CEA, carcinoembryonic antigen; I: ITGA2B, Integrin, Alpha 2b; SELP: P-selectin.
Figure 6
Figure 6
Diagnostic outcomes for platelet ITGA2B, and SELP mRNA in the NSCLC using ddPCR and RPL32 reference gene. (A) The absolute copy number of β-actin derived via ddPCR. (B) ROC curve for platelet ITGA2B, and SELP mRNA with NSCLC versus all controls in the subgroup of test cohort using ddPCR. (C) ROC curve for platelet ITGA2B, and SELP mRNA with NSCLC versus all controls in the subgroup of test cohort using RPL32 reference gene. ROC: receiver operating characteristics; NSCLC: non-small cell lung cancer; ddPCR: Droplet digital PCR; AUC: areas under the curves; ITGA2B: Integrin, Alpha 2b; SELP: P-selectin.
Figure 7
Figure 7
Prognostic prediction for OS according to platelets ITGA2B, platelets SELP, CEA and nomogram. Kaplan-Meier curves for OS according to platelets ITGA2B (A), platelets SELP (B) and CEA (C); (D) Nomogram model predicting 1-, and 2- year OS in NSCLC patients; The calibration curves for predicting patient OS at one year (E), and two years (F); (G) Decision curve analysis for 2-year survival predictions. OS: overall survival; ITGA2B: Integrin, Alpha 2b; SELP: P-selectin; CEA: carcinoembryonic antigen

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