hENT1 expression is down-regulated in pancreatic cancer tissues
The IHC results of 359 samples showed that the hENT1 protein was mainly localized in the membranes and/or the cytoplasm of cancer cells as previous literature described (Fig. 1a) 4. The average CES of hENT1 expression in tumor tissues was 80.5 and average CES in non-tumor tissues was 89.5. The hENT1 protein expression in tumor tissues was much lower than in normal tissues (Fig. 1b, 80.5 ± 8.8 versus 89.5 ± 8.9, p = 0.005). Subsequently, we use the average CES 80.5 as the cut-off value to divide 359 patients into hENT1 high expression group (n = 165) and low expression group (n = 194).
According to the grouping result, we then compared clinical pathological parameters between high and low hENT1 expression groups. The expression level of hENT1 was only related to the tumor differentiation degree, the patients in high hENT1 expression group tended to have highly differentiated tumors (Table 1). As to other pathological features such as gender, age, CA-199 level, tumor site, TNM stage, vascular and neural infiltration, they were not correlated with the expression level of hENT1.
Table 1
Relations between hENT1 expression and clinical characteristics
characteristics | Patients | hENT1 expression | |
n | % | High | Low | P value |
All patients | 359 | 100 | 165 | 194 | - |
Gender | 0.165 |
Male Female | 204 155 | 56.8 43.2 | 87 78 | 117 77 | |
Age | 0.432 |
< 65 >=65 | 239 120 | 66.6 33.4 | 106 59 | 133 61 | |
Diabetes | 0.758 |
Yes No | 48 308 | 13.4 85.8 | 21 144 | 27 164 | |
Chronic Pancreatitis | 1 |
Yes No | 1 358 | 0.3 99.7 | 0 165 | 1 193 | |
CA19-9,u/ml | 1 |
> 467 < 467 | 73 228 | 24.3 75.7 | 34 108 | 39 120 | |
Pathological differentiation degree | 0.032 |
High Low | 116 243 | 32.3 67.7 | 63 102 | 53 141 | |
Capsular invasion | 0.458 |
Yes No | 342 17 | 95.3 4.7 | 159 6 | 183 11 | |
Vascular infiltration | 0.167 |
是 否 | 28 331 | 7.8 92.2 | 9 156 | 19 175 | |
Neural infiltration | 0.525 |
Yes No | 165 194 | 46.0 54.0 | 79 86 | 86 108 | |
T classification | 0.653 |
T1/2 T3/4 | 21 338 | 5.8 94.2 | 11 154 | 10 184 | |
N classification | 0.336 |
N0 N1/2 | 153 206 | 42.6 57.4 | 75 90 | 78 116 | |
M classification | 1 |
M0 M1 | 336 23 | 93.6 6.4 | 154 11 | 182 12 | |
TNM stage | 0.574 |
I/II III/IV | 328 31 | 91.4 8.6 | 149 16 | 179 15 | |
Low hENT1 expression is correlated with a poor prognosis of gemcitabine-treated patients
To explore the relationship between the level of hENT1 and the prognosis of pancreatic cancer patients, firstly we analyzed the relationship between hENT1 expression level and disease free survival (DFS)/overall survival (OS) in all patients by Kaplan-Meier method and found a low hENT1 expression level indicated a significant poor outcome of PC patients, including shortened DFS(Fig. 2a, 21.6 ± 2.8 months versus 36.9 ± 4.0 months, p༜0.001) and OS(Fig. 2b, 33.6 ± 3.9 versus 39.6 ± 3.9, p = 0.004). Meanwhile, the result revealed that the level of CA19-9, the M stage, the TNM stage of patients and whether the tumor invaded into the pancreas capsule are also related to DFS when analyzed respectively. The DFS period of patients who had CA19-9 value ≤ 467 u/ml (the average value of all patients) were longer than those patients who had CA19-9 value༞467 u/ml preoperatively (Fig. 3f, 37.9 ± 4.1 versus 22.9 ± 4.0, p = 0.04). Similarly, patients in stage Ⅰ/Ⅱ of TNM stage had longer DFS compared with stage Ⅲ/Ⅳ patients (Fig. 3a, 31.0 ± 3.1 versus 12.4 ± 1.9, p = 0.016); as for M stage, patients in M0 stage had longer DFS than patients in M1 stage (Fig. 3c, 30.7 ± 3.0 versus 11.8 ± 2.2, p = 0.031); and patients with tumors not invading the capsule had better DFS than those with tumor invasion into the capsule (Fig. 3e, 30.8 ± 3.0 versus 12.6 ± 2.3, p = 0.053). What’s more, the M stage of patients and the TNM stage also had significant correlation with patients’ overall survival (OS). M0 stage patients had longer OS than M1 stage patients (Fig. 3d, 39.7 ± 3.4 versus 16.2 ± 1.9, p = 0.026); patients in stage Ⅰ/Ⅱ/of TNM stage had better OS than those in stage Ⅲ/Ⅳ (Fig. 3b, 40.2 ± 3.4 versus 15.4 ± 1.7,p = 0.002).
For further exploring the connection between hENT1 level and gemcitabine treatment efficacy, we analyzed the relationship between hENT1 expression and DFS/OS in two separated groups (patients in one group received gemcitabine treatment after surgery while the other group didn’t receive chemotherapy). The findings suggested that the results were much more remarkable in the gemcitabine subgroup, a high hENT1 expression level was related to longer DFS (Fig. 4a, 35.7 ± 4.0 versus 20.6 ± 2.7; p < 0.0001). Similarly, patients with high hENT1 expression in gemcitabine-treated group showed longer overall survival (OS) compared with low expression group (Fig. 4b, 39.4 ± 4.0 versus 31.5 ± 3.9, p = 0.001). In contrast, no significant difference of DFS and OS was found in non-gemcitabine treated group between the expression level of hENT1 and the prognosis of patients (Fig. 4c, d, p = 0.413 and p = 0.152).
A low hENT1 expression level is an independent indicator for poor prognosis of PC patients
Through the survival analysis, we found that the M stage, the TNM stage, whether or not have tumor capsular invasion, preoperative CA19-9 value and hENT1 expression level were connected with prognosis of pancreatic cancer patients. Therefore, we put these factors into multivariate analysis by COX regression to further evaluate their prognostic value in PC patients. The results showed that a low hENT1 expression level in tumor tissues was an independent risk factor for PC recurrence or metastasis, cause it was connected with shorter DFS in patients with pancreatic cancer (HR 0.53; 95% CI: 0.39–0.72; p༜0.001). In addition, an advanced TNM stage (HR 2.68; 95% CI: 1.09–6.58; P = 0.031) and a low expression level of hENT1 (HR 0.60; 95%CI: 0.43–0.82; p = 0.001) were independent risk factors for overall survival of PC (Table 2). Besides, we also put these factors into multivariate analysis in the separate gemcitabine-treated group and have produced similar findings, advanced TNM stage predicted worse OS (HR 2.90; 95%CI: 1.06–7.92; p = 0.038); low hENT1 expression level predicted worse OS (HR 0.60; 95%CI: 0.43–0.82; p = 0.002) and DFS (HR 0.56; 95%CI: 0.41–0.77; p༜0.001). In conclusion, low hENT1 expression level is an independent risk factor for patients’ shortened DFS and OS.
Table 2
In the general population, the correlations between several parameters and patients’ long-term prognosis.
Characteristics | OS (overall survival) | DFS (disease free survival) |
| Hazard ratio (HR) | 95% CI (confidence interval) | P value | Hazard Ratio (HR) | 95% CI (confidence Interval) | P value |
M classification | 0.65 | 0.22–1.91 | 0.433 | 0.91 | 0.32–2.64 | 0.867 |
| | | | | | |
TNM stage | 2.68 | 1.09–6.58 | 0.031 | 1.88 | 0.77–4.59 | 0.169 |
| | | | | | |
Capsular invasion | 0.84 | 0.37–1.92 | 0.682 | 0.74 | 0.34–1.59 | 0.440 |
| | | | | | |
CA19-9 | 1.19 | 0.85–1.67 | 0.309 | 1.36 | 0.99–1.86 | 0.057 |
| | | | | | |
hENT1 | 0.60 | 0.43–0.82 | 0.001 | 0.53 | 0.39–0.72 | ༜0.001 |
| | | | | | |
| | | | | | |
Table 3
In the gemcitabine-treated population, the correlations between several parameters and patients’ long-term prognosis.
Characteristics | OS (overall survival) | DFS (disease free survival) |
| Hazard ratio (HR) | 95% CI (confidence interval) | P value | Hazard Ratio (HR) | 95% CI (confidence Interval) | P value |
M classification | 1.03 | 0.32–3.36 | 0.963 | 0.98 | 0.30–3.19 | 0.976 |
| | | | | | |
TNM stage | 2.90 | 1.06–7.92 | 0.038 | 2.31 | 0.85–6.28 | 0.100 |
| | | | | | |
Capsular invasion | 0.87 | 0.38–1.98 | 0.734 | 0.76 | 0.35–1.64 | 0.485 |
| | | | | | |
CA19-9 | 1.16 | 0.82–1.65 | 0.395 | 1.32 | 0.95–1.83 | 0.097 |
| | | | | | |
hENT1 | 0.60 | 0.43–0.82 | 0.002 | 0.56 | 0.41–0.77 | ༜0.001 |