Unveiling diagnostic potential of extracellular DNA and lung tissue-specific X gene expression in non-small cell lung carcinoma patients

Background: Liquid biopsy (circulating cells and biomolecules) has revolutionized a non-invasive, efficient, and accurate alternative to tissue biopsy. Aim: To investigate the diagnostic utility of circulating cell-free (cf) DNA levels and lung tissue-specific X(LunX) gene expression and its association with micrometastasis in non-small cell lung carcinoma (NSCLC) patients. Methods: Blood (serum) samples of 81 NSCLC patients and matched 76 controls were collected, along with clinicopathological details. The cf-DNA was quantitated by amplifying β-globin and compared with the standard curve plotted by TaqMan control human genome DNA. LunX gene expression was measured by reverse transcription and SYBR Green chemistry-based real-time PCR. The relative fold was calculated by using the 2-^ΔΔCT method. Results: The mean cf. DNA levels in NSCLC, lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) were significantly higher compared to controls (p < 0.0001). LunX gene expression was significantly higher in NSCLC (2.04-fold, p < 0.0001) and LUSC (1.90-fold, p < 0.0001); however, it slightly decreased in LUAD (1.31-fold, p < 0.0001) patients. With increased tumor size (T3 and T4), cf. DNA levels significantly increased (p < 0.0001). However, TNM was not associated with cf. DNA levels (p < 0.05). In contrast, LunX gene expression showed a higher fold with involvement of lymph nodes (N2; p = 0.015, N3; p = 0.009) and LunX 2-fold higher expression of LunX in metastasis (p = 0.0001). Smoking pack-year significantly influenced the levels of cf. DNA and LunX gene expression (p < 0.0001). There was no correlation between cfDNA levels and LunX gene expression (r = 0.06, p = 0.63). LunX demonstrated superior performance (AUC;0.886) with high sensitivity (100%) and specificity (62.96%). The cfDNA also showed good accuracy (AUC;0.729) but had a relatively low PPV. Conclusion: LunX and cfDNA hold promise as potential non-invasive diagnostic biomarkers for NSCLC; however, LunX exhibited superior diagnostic performance in this study.