Patient characteristics
A total of 97 patients diagnosed with LUAD were enrolled from March 2014 to August 2015. There were 62 (63.9%) males and 35 females (36.1%) with median age 58 years (31–77 years). In addition, 35 patients (36.1%) had a history of smoking, and 52 (53.6%) had lymph node metastasis, and 36 (37.1%) harbored larger tumors (> 4 cm). There were 34 (35.1%), 23 (23.7%), 26 (26.8%) and 14 (14.4%) patients with stage I, II, III and IV tumors respectively. Forty-four patients (45.4%) had moderately differentiated tumors, 12 (12.4%) had well-differentiated tumors, and 41 (42.3%) had poorly differentiated tumors.
CTC counts and correlation to patient status and pathological features
The CTCs of different phenotypes are shown in Fig. 1A-C. The positive rate of CTCs was 96.91% (0 to 73) in the entire cohort, the median of CTCs was 6 and the average of CTCs values was 10.29 ± 13.43 (Table 1). The number of CTCs increased with tumor stage progression (P = 0.0340 between stage I and IV, Fig. 2A). The number and positive rate of M-CTC also increased with the stage, and were respectively 41.18%, 65.22%, 76.92% and 78.57% in stages I, II, III and IV (P = 0.0133 between stage I and II, P = 0.0015 between stage I and III, P = 0.0026 between stage I and IV, Fig. 2B). However, the frequencies of E/M-CTC and E-CTC were not affected by LUAD progression. In addition, M-CTC showed significant correlation with lymphatic metastasis (P < 0.001, OR = 5.100, 95% CI = 2.094–12.426, Table 2) and stage (P = 0.009, OR = 3.326, 95% CI = 1.344–8.227, Table 2).
Table 1
Positive expression rate of CTCs in each NSCLC stage n(%)
Stating | Numbers | CTCs | E/M-CTCs | E-CTCs | M-CTCs | Median CTCs | CTCs average | CTCs range |
I | 34 | 32(94.12) | 27(79.41) | 20(58.82) | 14(41.18) | 4.00 | 7.21 | 0–39 |
II | 23 | 22(95.65) | 17(73.91) | 16(69.57) | 15(65.22) | 6.00 | 11.70 | 0–68 |
III | 26 | 26(100.00) | 23(88.46) | 8(30.77) | 20(76.92) | 7.50 | 10.23 | 1–49 |
IV | 14 | 14(100.00) | 12(85.71) | 8(57.14) | 11(78.57) | 13.50 | 15.57 | 1–73 |
total | 97 | 94(96.91) | 79(81.44) | 52(53.61) | 60(61.86) | 6.00 | 10.29 | 0–73 |
Abbreviations: CTC, circulating tumor cell; M-CTC, CTCs with epithelial-mesenchymal transition phenotype; E-CTC, CTCs with epithelial phenotype; E/M-CTC, CTCs with mixed phenotypes; OR ,risk ratio; CI ,confidence interval. |
Table 2
Association between clinical parameters and M-CTC
Group | | M-CTC positive | OR(95%CI) | P-Value |
n | n | % |
Gender | | | | | | |
Female | 35 | 20 | 57.14 | 1.364 (0.584–3.184) | 0.473 |
Male | 62 | 40 | 64.52 | | | |
Age | | | | | | |
≤65 | 72 | 47 | 65.28 | 0.578(0.229–1.450) | 0.242 |
༞65 | 25 | 13 | 52.00 | | | |
Smoking | | | | | | |
No | 62 | 36 | 58.06 | 1.576(0.858–3.776) | 0.308 |
Yes | 35 | 24 | 68.57 | | | |
Lymphatic metastasis | | | | | | |
N- | 45 | 19 | 42.22 | 5.100(2.094–12.426) | ༜0.001 |
N+ | 52 | 41 | 78.85 | | | |
Tumor Size, cm | | | | | | |
≤ 4 | 61 | 35 | 57.38 | 1.688(0.706–4.038) | 0.239 |
༞4 | 36 | 25 | 69.44 | | | |
Stage | | | | | | |
I + II | 57 | 29 | 50.88 | 3.326(1.344–8.227) | 0.009 |
II + IV | 40 | 31 | 75.50 | | | |
Differentiated degree | | | | | | |
Moderately + Well | 56 | 32 | 57.14 | 1.615(0.694–3.758) | 0.266 |
Poorly | 41 | 28 | 68.29 | | | |
Note: Bold values indicate statistically significant values. |
Abbreviations: CTC, circulating tumor cell; M-CTC, CTCs with epithelial-mesenchymal transition phenotype; OR ,risk ratio; CI ,confidence interval. |
Napsin A expression and correlation with patient status and pathological features
Twenty patients (20.62%) did not express Napsin A in the tumor tissues (Fig. 3). As shown in Table 3, Napsin A expression correlated significantly to lymphatic metastasis (P = 0.004, OR = 0.147, 95%CI = 0.040–0.543) but not with gender, age, tumor size, stage, differentiated degree and smoking. In addition, the Napsin A- patients had a greater frequency of M-CTC compared to the Napsin A + patients (Fig. 4). The positive rate of M-CTC was also higher in the Napsin A- versus Napsin A + patients (P = 0.010, OR = 0.133, 95%CI = 0.028–0.620), whereas that of E/M-CTC and E-CTC were not significantly different between the two groups (Table 4).
Table 3
Association between clinical parameters and Napsin A
Group | | Napsin A positive | OR(95%CI) | P-Value |
n | n | % |
Gender | | | | | | |
Female | 35 | 28 | 80.00 | 0.942(0.337–2.638) | 0.910 |
Male | 62 | 49 | 79.03 | | | |
Age | | | | | |
≤65 | 72 | 55 | 76.39 | 2.267(0.604–8.512) | 0.225 |
༞65 | 25 | 22 | 88.00 | | | |
Smoking | | | | | | |
Yes | 62 | 51 | 82.26 | 0.623(0.229–1.693) | 0.354 |
No | 35 | 26 | 74.29 | | | |
Lymphatic metastasis | | | | | | |
N- | 45 | 35 | 77.78 | 0.147(0.040–0.543) | 0.004 |
N+ | 52 | 42 | 80.77 | | | |
Tumor Size, cm | | | | | | |
≤ 4 | 61 | 48 | 78.69 | 1.122(0.401–3.137) | 0.826 |
༞4 | 36 | 29 | 80.56 | | | |
Stage | | | | | | |
I + II | 57 | 47 | 82.46 | 0.638(0.237–1.716_ | 0.374 |
II + IV | 40 | 30 | 75.00 | | | |
Differentiated degree | | | | | | |
Moderately + Well | 56 | 42 | 75.00 | 1.944(0.676–5.592) | 0.217 |
Poorly | 41 | 35 | 85.37 | | | |
Note: Bold values indicate statistically significant values. |
Table 4
Association between CTCs and Napsin A
| Napsin A | | |
Group | Positive(n) | Negative(n) | OR(95%CI) | P-Value |
E/M-cells | | | | |
(+) | 61 | 18 | 0.487(0.095–2.489) | 0.487 |
(-) | 16 | 2 | | |
M-Cells | | | | |
(+) | 42 | 18 | 0.133(0.028–0.620) | 0.010 |
(-) | 35 | 2 | | |
E-CTC | | | | |
(+) | 40 | 12 | 0.712(0.247–2.054) | 0.530 |
(-) | 37 | 8 | | |
Note: Bold values indicate statistically significant values. |
Abbreviation: CTC, circulating tumor cell; M-CTC, CTCs with epithelial-mesenchymal transition phenotype; E-CTC, CTCs with epithelial phenotype; E/M-CTC, CTCs with epithelial/mesenchymal phenotype; OR ,risk ratio; CI ,confidence interval. |
Prognostic Significance Of M-ctcs And Napsin A In Luad
All patients were followed up for at least 60 months, during which 73 (75.26%) patients relapsed and 59 (60.82%) died.
The M-CTC + patients had worse RFS (P < 0.0001, Fig. 5A) and OS (P < 0.0001, Fig. 5B) compared to the M-CTC- patients. Likewise, the Napsin A- patients also showed worse RFS (P < 0.0001, Fig. 6A) and OS (P = 0.0003, Fig. 6B) compared to the Napsin + group. The patients are divided into the M-CTC+/Napsin A- (18/97, 18.56%), M-CTC-/Napsin A- (2/97, 2.06%), M-CTC+/Napsin A+ (42/97, 43.3%) and M-CTC-/Napsin A+ (35/97, 36.08%) subgroups, of which M-CTC+/Napsin A- patients had the worst RFS (P < 0.0001, Fig. 7A) and OS (P < 0.0001, Fig. 7B).
Univariate analysis (Table 5) showed that gender (P = 0.032, HR = 1.697. 95% CI = 1.034–2.785), smoking (P = 0.035, HR = 1.660, 95% CI = 1.036–2.659), M-CTC (P༜0.001, HR = 2.866, 95%CI = 1.722–4.771), lymph node metastasis (P < 0.001, HR = 3.377, 95%CI = 2.054–5.553), tumor size (P = 0.007, HR = 1.904, 95%CI = 1.192–3.042), stage (P = 0.007, HR = 1.905, 95%CI = 1.196–3.035), Napsin A (P༜0.001, HR = 0.321, 95%CI = 0.186–0.555) and degree of differentiation (P = 0.010, HR = 1.850, 95%CI = 1.162–2.946) was significantly correlated to RFS. OS was influenced by gender (P = 0.041, HR = 1.803. 95% CI = 1.026–3.171), smoking (P = 0.004, HR = 2.146, 95% CI = 1.282–3.591), M-CTC (P < 0.001, HR = 3.289, 95%CI = 1.798–6.014), lymph node metastasis (P < 0.001, HR = 2.681, 95%CI = 1.557–4.620), tumor size (P = 0.002, HR = 2.253, 95%CI = 1.347–3.769), stage (P < 0.001, HR = 2.530, 95%CI = 1.510–4.238), Napsin A (P < 0.001, HR = 0.364, 95%CI = 0.206–0.643) and degree of differentiation (P = 0.004, HR = 2.115, 95%CI = 1.262–3.545). According to the multivariate analysis (Table 6), We found that the RFS of patients with M-CTC positive was shorter than that of patients with M-CTC negative, and the difference was statistically significant (P = 0.009, HR = 2.105, 95%CI = 1.206–3.676), and the RFS of patients with negative expression of Napsin A was shorter than that of patients with positive, and the difference was statistically significant (P = 0.032, HR = 0.507, 95%CI = 0.272–0.943). In addition, M-CTC-positive patients had shorter overall survival than negative patients (P = 0.010, HR = 2.319, 95%CI = 1.218–4.418), and patients with negative expression of Napsin A had worse overall survival (P = 0.046, HR = 0.504, 95%CI = 0.257–0.988). Therefore, the results of these studies indicate that positive expression of M-CTC and negative expression of Napsin A can indicate poor prognosis in patients with LUAD, and can be used as A potential marker for diagnosis, identification and prognosis evaluation of lung adenocarcinoma. Based on the multivariate Cox proportional hazard regression model, we established a nomogram on the OS and RFS of LUAD (Fig. 8A-B).
Table 5
Univariate analysis for recurrence-free survival and overall survival
Variable | Level | RFS | OS |
---|
| | HR (95% CI) | P-value | HR (95% CI) | P-value |
Gender | female/male | 1.697(1.034–2.785) | 0.032 | 1.803(1.026–3.171) | 0.041 |
Age | ≤ 65/༞65 | 1.078(0.639–1.818) | 0.779 | 1.215(0.684–2.158) | 0.506 |
Smoking | Yes/No | 1.660(1.036–2.659) | 0.035 | 2.146(1.282–3.591) | 0.004 |
M-CTC | Yes/Not | 2.866(1.722–4.771) | ༜0.001 | 3.289(1.798–6.014) | ༜0.001 |
Lymphatic metastasis | N0/N+ | 3.377(2.054–5.553) | ༜0.001 | 2.681(1.557–4.620) | ༜0.001 |
Tumor Size, cm | ≤ 4/༞4 | 1.904(1.192–3.042) | 0.007 | 2.253(1.347–3.769) | 0.002 |
Stage | I + II/III + IV | 1.905(1.196–3.035) | 0.007 | 2.530(1.510–4.238) | ༜0.001 |
Napsin A | Negative/Positive | 0.321(0.186–0.555) | ༜0.001 | 0.364(0.206–0.643) | ༜0.001 |
Differentiated degree | Moderately + Well / Poorly | 1.850(1.162–2.946) | 0.010 | 2.115(1.262–3.545) | 0.004 |
Note: Bold values indicate statistically significant values. |
Abbreviation: CTC, circulating tumor cell; M-CTC, CTCs with epithelial-mesenchymal transition phenotype; HR, hazard ratio; CI ,confidence interval ; RFS, Recurrence-free survival; OS, Overall survival. |
Table 6
Multivariate analysis for recurrence-free survival and overall survival
Variable | RFS | OS |
---|
| HR ∗ (95% CI) | P-value∗ | HR∗ (95% CI) | P-value∗ |
M-CTC | | | | |
(+) | 2.105(1.206–3.676) | 0.009 | 2.319(1.218–4.418) | 0.010 |
(-) | | | | |
Napsin A | | | | |
Positive | 0.507(0.272–0.943) | 0.032 | 0.504(0.257–0.988) | 0.046 |
Negative | | | | |
Notes: ∗HR and P-value for Cox proportional hazard regression model. adjustment by Gender, Smoking,Lymphatic metastasis༌Tumor Size༌Stage༌Differentiated degree. Bold values indicate statistically significant values. |
Abbreviation:M-CTC, CTCs with epithelial-mesenchymal transition phenotype; HR, hazard ratio; CI ,confidence interval ; RFS, Recurrence-free survival; OS, Overall surviva. |