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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

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.


Assessment of platelet purity
To assess sample purity, three freshly isolated and randomly selected platelet isolations in RNAlater were fixed in 3.7% paraformaldehyde and counted by the Sysmex XN2000 haematology analyser and stained by Wright-Gimsa. The platelet morphology was confirmed on a light microscope by two observers (Xing S and Zeng T), which displayed full of the vision with 0-1 other cell ( Figure S1A, 200×；Figure S1B, 400×). Total platelet and nucleated cell counts was determined by the Sysmex XN2000 haematology analyser in both the sheath flow DC detection( Figure S1C) and PLT-fluorescent method ( Figure S1D) and yielded an estimated 1 to 5 nucleated cell counts per 10 million platelets, which is in concordance to observations by others[1].

The primers of the candidate TEP mRNA
The following primers were used: BSG, forward primer: 5 ′ -  Figure S2.

The protein expression levels of platelet ITGA2B and SELP
We detected the protein expression levels of platelet ITGA2B and SELP. The antibodies for Flow Cytometry (FC) and western blotting (WB) was purchased from R&D system, USA. FC was performed using a BD FACSCalibur flow cytometer. All the experiments were done following the manufacturer's instructions. As shown in the Figure S4, FC results showed that no difference between NSCLC group and HC group was found. We also conducted western blotting. In brief, platelet proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Then, they are incubated with antibodies to ITGA2B (2ug/ml, MAB7616, R&D), SELP (1ug/ml, AF137, R&D), and α-tubulin (1:3,000, ab9781, Abcam) at 4°C overnight, then treated with a horseradish peroxidase-conjugated secondary antibody. The results were similar to FC.

The expression and diagnostic analysis of PLT, MPV and IPF
We utilized whole blood samples from the test cohort and validation cohort to estimate the discriminatory capability of PLT and MPV. 18 patients of NSCLC and 11 healthy controls were also introduced to evaluate the diagnostic effect of IPF absolute value (IPF#) and IPF fraction (IPF%). As shown in the Figure

The diagnostic performance of platelet ITGA2B in subgroups of NSCLC
CEA is the most common used tumor markers in NSCLC. However, its sensitivity is low, especially at the stage I stage. We tried to evaluate whether ITGA2B had supplementary diagnostic value for this CEA negative subgroup of NSCLC. ROC curves were plotted for platelet ITGA2B in those CEA-negative NSCLC patients versus different control groups, seen in the Figure S6. In the detection of CEA-negative NSCLC patients from all control subjects (HC and BPN), the AUC of ITGA2B was 0.951 (95% CI 0.907-0.996) with a sensitivity of 97.8% and specificity of 78.6%. In the differentiation of CEA-negative NSCLC from the BPN, the similar result was found. In addition, the diagnostic values of platelet ITGA2B in CEA-positive NSCLC were also investigated. The ROC curves shown in Figure S6 E-H indicated that platelet ITGA2B could distinguish CEA-positive NSCLC from noncancerous population. Simultaneously, the validation cohort verified the above diagnostic significance of platelet ITGA2B. and specificity: 76.2%), the results were similar in the validation cohort, seen in the Table S4 and Figure S7.  Figure S8E, 8G showed that platelet ITGA2B mRNA could discriminate CEA-positive stage I NSCLC from noncancerous people. Similar results were obtained in the validation cohort.

The diagnostic performance of platelet SELP in validation cohort
In detection of NSCLC from all control subjects (HC and BPN), the AUC of SELP was 0.716 (95% CI 0. 616-0.815, Figure S9) with a sensitivity of 96.7% and specificity of 43.1%. In differentiation of NSCLC from BPN, the sensitivity was the same (96.7%) with a specificity of 43.8%. In the differentiation of stage I NSCLC from the BPN, the similar result was found.

The diagnostic performance of platelet ITGA2B in ADC or SqCC
As shown in Figure S10, the values of platelet ITGA2B did not differ significantly between the two groups in both cohorts. ROC  Table S1 Steps in refining the biomarker candidates TEPs refinement steps Figure  1220 up and 570 down number of DEGs Figure 2A 208 overlappped reproducibilitity in GSE68086 and 89843 Figure 2D 148 upregulated upregulated DEGs 119 upregulated in at least 7/ 9 NSCLC sensitivity 104 RPKM > 20 easier to be detected 8 core DEGs functionnally and previous report