NDRG2 was downregulated in patients with LUAD compared with that in normal tissues
To investigate whether NDRG2 was detectable and altered in LUAD patients compared with normal tissues, immunohistochemistry (Figure 1 A-C), Western blotting (Figure 1 D), and RT-PCR (Figure 1 E) were carried out to detect the expression levels of NDRG2. NDRG2 protein was mainly appeared in the cytoplasm, and a weak expression could be found in a limited number of cell nuclei. The results showed that the expression of NDRG2 at protein and mRNA levels in LUAD was significantly decreased compared with normal tissues.
Relationship between expression level of NDRG2 and clinicopathological features of patients with LUAD
As shown in Figure 2, the expression level of NDRG2 was remarkably correlated with CEA (P < 0.001).
To assess the correlation between the expression level of NDRG2 and clinical data, clinicopathological features of patients with LUAD are summarized in Table 2. The chi-square test revealed that the expression level of NDRG2 was notably higher in LUAD tissues in stages I–II than that in stage III–IV (P<0.001). In addition, the incidence of no vascular invasion and EGFR positive (+) was significantly higher in patients with high expression level of NDRG2 than that in patients with low expression level of NDRG2 (P<0.001 and 0.001, respectively). However, there were no associations between expression levels of NDRG2 and other clinicopathological features, including patients’ age, gender, smoking history, and blood type (P>0.05).
Regarding 34 patients who received surgery, the expression level of NDRG2 was significantly higher in stage I–II than that in stage III–IV (P=0.028). The incidence of no vascular invasion and EGFR positive (+) was markedly higher in patients with high expression level of NDRG2 than that in the patients with low expression level of NDRG2 (0.008 and 0.030, respectively).
Prognostic implications of NDRG2 and EGFR expression
Based on the clinicopathologic features of LUAD patients, as well as the expression levels of NDRG2, EGFR, and CEA, the survival time was analyzed by Kaplan–Meier method (Figure 3 A-H). The results showed that iodine-125 radioactive seeds brachytherapy for advanced LUAD patients with high expression level of NDRG2 had significantly higher OS than LUAD patients with low expression level (P = 0.0261, Figure 3A); besides, LAUD patients with EGFR(+) and CEA < 2.0 ng/ml had higher OS (P < 0.0001, 0.0314, Figure 3 B, C). Additionally, in operated patients with high expression level of NDRG2 (Figure 3E), EGFR(+) (Figure 3F), and CEA < 2.0 ng/ml (Figure 3G), higher OS was noted (P = 0.0022, < 0.0001 and 0.013, respectively).
According to the conjoined expressions of NDRG2/EGFR, the subjects were categorized into four groups: NDRG2-low/EGFR-negative(-), NDRG2-low/EGFR-positive(+), NDRG2-high/EGFR-negative(-), and NDRG2-high/EGFR-positive(+). The association between co-expression of NDRG2/EGFR and the OS was tested by Kaplan–Meier method. In these four groups, iodine-125 radioactive seeds brachytherapy for advanced LUAD patients in NDRG2-high/EGFR(+) group accompanied by the best prognosis during the 5-year follow-up period (P < 0.0001, Figure 3D), and the same results were observed in operated patients (P = 0.0002, Figure 3H).
Cox regression analysis
As shown in Table 3, NDRG2-low/EGFR(+) (hazard ratio (HR)=6.508; 95% confidence interval (CI), 2.619‑16.174; P<0.001), NDRG2-high/EGFR(+) (HR=3.519; 95% CI, 1.384‑8.949; P=0.008), and vascular invasion (HR=4.480; 95%CI, 2.291‑8.760; P<0.001) were independent prognostic factors of OS.