Low hENT1 expression indicates poor prognosis in gemcitabine-treated pancreatic cancer patients

Background: Pancreatic cancer (PC) is still a lethal disease and has a poor prognosis, gemcitabine-based chemotherapy is now the standard regimen in the treatment of pancreatic cancer. Gemcitabine resistance is an important obstacle for effective treatment of patients and improvement in patients’ overall survival (OS) and disease free survival (DFS). Results: Current research shows that human equilibrative nucleoside transporter 1 (hENT1) is related to gemcitabine chemoresistance for it mediates drug entry into cancer cells, and has an aberrant expression level in tumor tissues. But now discrepancies still exist between different research groups and studies about the expression level and prognostic value of hENT1 in PC patients, and the exactly underlying mechanism through which hENT1 transfer GEM into tumor cells remains unclear. Conclusion: hENT1 was low expressed in tumor tissues and this decreased expression level indicated a worse outcome (including shortened OS and DFS) in patients who received gemcitabine treatment postoperatively.

. Given this, finding a novel effective biomarker to identify patients those who are sensitive to gemcitabine and to predict the outcome of patients is imperative.
Human equilibrative nucleoside transporter 1 (hENT1), which was reported to facilitate cross-membrane transport of nucleosides and nucleoside-derived drugs, plays an important role in cancer chemotherapy 4 . Most notably, an elevated hENT1 expression is regarded as a diagnostic and therapeutic biomarker for PC patients treated with gemcitabine 5 − 7 . High hENT1 expression was also associated with better survival in other kinds of cancer patients who received gemcitabine treatment such as cholangiocarcinoma 8,9 , leiomyosarcoma and angiosarcoma 10 . But some studies reached an opposite conclusion that hENT1 expression level had no relation with patients' outcome 11,12 . Thus the prognostic value of hENT1 in PC patients still needs to be further verified by large sample size studies.
Here we performed immunohistochemistry (IHC) to assess hENT1 expression levels in 359 tumor tissues and adjacent normal tissues from surgical specimens of PC patients, and analyzed the relationships between hENT1 expression level and several clinicopathological features. We found that tumor tissues tended to have a relative low hENT1 expression level and this low expression level was correlated with tumor differentiation degree rather than other clinical parameters. In addition, we evaluated the prognostic value of hENT1 and drew the conclusion that a low hENT1 expression level is an independent risk factor for gemcitabine-treated patients.

Tissue sample collection
We collected the surgical pathological tissue of pancreatic cancer patients who received radical surgery in our hospital from September 2004 to December 2014   continuously, the eligible criteria for patients included having R0 surgical resection,   postoperative pathology diagnosis were pancreatic ductal adenocarcinoma, having no gemcitabine-based neoadjuvant chemotherapy and/or radiotherapy before surgery and surgical specimens were suitable for immunohistochemistry. In addition, these patients started adjuvant chemotherapy within 8 weeks after surgery with a regular follow-up monitoring CA-199/CT/B-ultrasonography and their clinical and pathological data were complete. We excluded patients who had severe basic diseases or had serious complications during perioperative period which could affect survival analysis results, and those who had poor compliance and cannot being followed regularly. We finally had 375 samples for the next-step analysis. This study was approved by the Ethics Committee of Beijing Union Medical College Hospital. 375 pancreatic cancer tissues and paired adjacent non-tumor tissues were employed for the construction of 4 tissue microarrays (TMAs) using routine methods, namely TMA1, TMA2, TMA3, TMA4. The quality of 4 TMAs was reexamined by HE staining and in accordance with the design requirements. We did immunohistochemistry analysis to test hENT1 expression and excluded 16 samples because the tissue sections detached from the slides. The hENT1 antibody was used to measure hENT1 expression in TMAs by IHC staining according to standard protocols. TMAs were firstly blocked by hydrogen peroxide and then incubated with an anti-hENT1 antibody (1:200, Anti-SLC29A1 polyclonal antibody produced in rabbit from Sigma-Alorich Company in America). Subsequently, DAB (diaminobezidin) was used for coloration and hematoxylin was used for counterstaining. All immunohistochemistry results were determined by two independent pathologists on a double-blind basis.
The staining intensities were graded as 0 (negative), 1 (low), 2 (medium), or 3 (high) while the staining extent was scored from 0 to 100%. And intensity score × percentage score × 100 made up the final IHC staining score together, namely composite expression score (CES), which ranged from 0 to 300. The average CES value of 359 tumor tissues 80.5 was defined as the optimal cutoff value. CES 80.

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. 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, Table 3 In the gemcitabine-treated population, the correlations between several parameters and patients' long-term prognosis. extensively. Given the current situation that gemcitabine is the preferred postoperative chemotherapy drug, using hENT1 as an index to distinguish those who are sensitive to gemcitabine and predict the long-term prognosis of patients is very promising.

Conflicts of Interests:
None declared.

Ethics approval and consent to participate
All participants provided written informed consent. Patient data were de-identified and anonymized before analysis. The study on human data collection was approved by the Ethics Committee of the Peking Union hospital.

Availability of data and materials
All data generated or analyzed during this study are included in this published article. Several clinical parameters significantly related to patients' prognosis. a, b, patients in I/II T Figure 4 hENT1 expression level is related to patients' prognosis in gemcitabine-treated group. a, b, i