The prognostic and predictive role of class III ß-Tubulin and hENT1 expression in patients with advanced pancreatic ductal adenocarcinoma.

BACKGROUND: FOLFIRINOX and gemcitabine-nabpaclitaxel (GnP) are standard first-line treatment regimens for advanced pancreatic ductal adenocarcinoma (PDAC). However, currently, there is a lack of predictive biomarkers to aid in the treatment selection. We aimed to explore the prognostic and predictive value of class III ß-Tubulin (TUBB3) and human equilibrative nucleoside transporter 1 (hENT1) expression, which have previously been shown to be associated with taxane and gemcitabine resistance in advanced PDAC. METHODS: We conducted a retrospective analysis of 106 patients with advanced PDAC treated with GnP and/or FOLFIRINOX at our institution. TUBB3 and hENT1 immunohistochemical staining was performed on tumor specimens and subsequently evaluated based on the intensity and percentage of expression. RESULTS: In patients who received the GnP regimen, a high combined score (TUBB3low/hENT1high) was associated with a higher DCR and longer PFS compared to those with intermediate (TUBB3high/hENT1high or TUBB3low/hENT1low) and low score (TUBB3high/hENT1low). In the multivariate analysis, a high combined score was an independent predictor of higher DCR (OR:11.96; 95 % CI:2.61-54.82; p = 0.001) and longer PFS (HR:0.33; 95%CI:0.18-0.60; p < 0.001). However, there was no difference in response rates or PFS based on TUBB3 and hENT1 expression among patients receiving the FOLFIRINOX regimen. CONCLUSION: Our findings indicate that tumor TUBB3 and hENT1 expression may predict the efficacy of the GnP regimen, and low TUBB3 and high hENT1 expression (TUBB3low/hENT1high) are associated with a higher DCR and longer PFS in patients treated with GnP. Evaluating TUBB3 and hENT1 jointly can identify the patients most (as well as least) likely to benefit from GnP chemotherapy.

Concordance of human equilibrative nucleoside transporter-1 expressions between murine (10D7G2) and rabbit (SP120) antibodies and association with clinical outcomes of adjuvant chemotherapy for pancreatic cancer: A collaborative study from the JASPAC 01 trial.

BACKGROUND: Expression of human equilibrative nucleoside transporter-1 (hENT1) is reported to predict survival of gemcitabine (GEM)-treated patients. However, predictive values of immunohistochemical hENT1 expression may differ according to the antibodies, 10D7G2 and SP120. AIM: We aimed to investigate the concordance of immunohistochemical hENT1 expression between the two antibodies and prognosis. METHODS: The subjects of this study were totally 332 whose formalin-fixed paraffin-embedded specimens and/or unstained sections were obtained. The individual H-scores and four classifications according to the staining intensity were applied for the evaluation of hENT1 expression by 10D7G2 and SP120, respectively. RESULTS: The highest concordance rate (79.8%) was obtained when the cut-off between high and low hENT1 expression using SP120 was set between moderate and strong. There were no correlations of hENT1 mRNA level with H-score (p = .258). Although the hENT1 mRNA level was significantly different among four classifications using SP120 (p = .011), there was no linear relationship among them. Multivariate analyses showed that adjuvant GEM was a significant predictor of the patients with low hENT1 expression using either 10D7G2 (Hazard ratio [HR] 2.39, p = .001) or SP120 (HR 1.84, p < .001). In contrast, agent for adjuvant chemotherapy was not significant predictor for the patients with high hENT1 expression regardless of the kind of antibody. CONCLUSION: The present study suggests that the two antibodies for evaluating hENT1 expression are equivalent depending on the cut-off point and suggests that S-1 is the first choice of adjuvant chemotherapy for pancreatic cancer with low hENT1 expression, whereas either S-1 or GEM can be introduced for the pancreatic cancer with high hENT1 expression, no matter which antibody is used.

Membrane Localization of Human Equilibrative Nucleoside Transporter 1 in Tumor Cells May Predict Response to Adjuvant Gemcitabine in Resected Cholangiocarcinoma Patients.

BACKGROUND: The use of gemcitabine as an adjuvant modality for cholangiocarcinoma (CC) is increasing, but limited data are available on predictive biomarkers of response. Human equilibrative nucleoside transporter 1 (hENT-1) is the major transporter involved in gemcitabine intracellular uptake. This study investigated the putative predictive role of hENT-1 localization in tumor cells of CC patients undergoing treatment with adjuvant gemcitabine. METHODS: Seventy-one consecutive patients with resected CC receiving adjuvant gemcitabine at our center were retrospectively analyzed by immunohistochemistry for hENT-1 localization in tumor cells. The main outcome measure was disease-free survival (DFS). Hazard ratios (HRs) of relapse and associated 95% confidence intervals (CIs) were obtained from proportional hazards regression models stratified on quintiles of propensity score. RESULTS: Twenty-three (32.4%) cases were negative for hENT-1, 22 (31.0%) were positive in the cytoplasm only, and 26 (36.6%) showed concomitant cytoplasm/membrane staining. Patients with membrane hENT-1 had a longer DFS (HR 0.49, 95% CI 0.24-0.99, p = .046) than those who were negative or positive only in the cytoplasm of tumor cells. Notably, the association between DFS and membrane hENT-1 was dependent on the number of gemcitabine cycles (one to two cycles: HR 0.96, 95% CI 0.34-2.68; three to four cycles: HR 0.99, 95% CI 0.34-2.90; five to six cycles: HR 0.27, 95% CI 0.10-0.77). CONCLUSION: hENT-1 localization on tumor cell membrane may predict response to adjuvant gemcitabine in CC patients receiving more than four cycles of chemotherapy. Further prospective randomized trials on larger populations are required to confirm these preliminary results, so that optimal gemcitabine-based chemotherapy may be tailored for CC patients in the adjuvant setting. IMPLICATIONS FOR PRACTICE: Gemcitabine is becoming an increasingly used adjuvant modality in cholangiocarcinoma (CC), but limited data are available on predictive biomarkers of response. In this study, patients receiving more than four cycles of adjuvant gemcitabine and harboring Human equilibrative nucleoside transporter 1 (hENT-1, the major transporter involved in gemcitabine intracellular uptake) on tumor cell membrane had a longer disease-free survival compared with patients negative or positive for hENT-1 only in the cytoplasm of tumor cells. Overall these results may lay the basis for further prospective randomized trials based on a larger population of patients and may prove useful for tailoring appropriate gemcitabine-based chemotherapy for CC patients in the adjuvant setting.

Prognostic Value of Excision Repair Cross-Complementing Gene 1, Dihydropyrimidine Dehydrogenase, and Human Equilibrative Nucleotide Transporter 1 Expression and Their Implications for Adjuvant Treatment in Patients With Ampullary Carcinoma.

OBJECTIVES: The purpose of this study was to characterize the intratumoral expression profiles of excision repair cross-complementing gene 1 (ERCC1), dihydropyrimidine dehydrogenase (DPD), and human equilibrative nucleotide transporter 1 (hENT1) in ampullary carcinomas (ACs) to evaluate their prognostic values and better tailor adjuvant chemotherapy for individual patients with AC after surgery. METHODS: This study included 49 patients with AC who underwent a curative pancreaticoduodenectomy. Various clinicopathological factors, including ERCC1, DPD, and hENT1, were analyzed in relation to postoperative disease recurrence and the patients' survival. RESULTS: The median recurrence-free survival and overall survival were 24.5 months and 32.4 months, respectively. Multivariate Cox regression analysis of recurrence-free survival identified a DPD expression (hazard ratio [HR], 8.18; 95% confidence interval [CI], 2.00-34.8; P = 0.003) and combined ERCC1/DPD expression (HR, 134.8; 95% CI, 11.8-1920; P < 0.001) as independent predictors of disease recurrence. Multivariate Cox regression analysis of overall survival also identified a DPD expression (HR, 8.48; 95% CI, 1.71-46.3; P = 0.008) and combined ERCC1/ DPD expression (HR, 135.6; 95% CI, 11.8-1940; P < 0.001) as independent predictors of survival. CONCLUSIONS: The DPD and ERCC1 expression profile could potentially serve as a useful prognostic biomarker and therapeutic target for surgically resected patients with AC.

Human equilibrative nucleoside transporter 1 testing in pancreatic ductal adenocarcinoma: a comparison between murine and rabbit antibodies.

AIMS: The human equilibrative nucleoside transporter 1 (hENT1) expression level in pancreatic ductal adenocarcinoma (PDAC) may predict survival in gemcitabine-treated patients after resection. These results have been obtained with a murine anti-hENT1 antibody (10D7G2) that is not commercially available. Another antibody, which is rabbit-derived (SP120), appears to have no predictive value in local, advanced or metastatic PDAC. We aimed to study whether the two antibodies are equivalent. METHODS AND RESULTS: We compared hENT1 expression with both antibodies in resected PDAC. The results were correlated with overall survival (OS) following gemcitabine treatment. Tissues from two sets of patients (n = 147 each) were stained with SP120 by the use of different equipment, with an amplification technique being used for set 2. The rate of 'hENT1 high' cases was lower with SP120 (set 1, 7% versus 48%; set 2, 11% versus 38%). With the amplification technique, the rate of hENT1 high cases was globally similar between both antibodies. However, concordance between the antibodies was found in only 50% of cases. High hENT1 expression was predictive of OS only with 10D7G2 (hazard ratio 0.49; 95% confidence interval 0.24-0.98; P = 0.045). CONCLUSIONS: The two antibodies are not equivalent. Further prospective studies seem to be warranted before hENT1 testing for PDAC is used in daily practise.

ENT1 inhibition attenuates epileptic seizure severity via regulation of glutamatergic neurotransmission.

Type 1 equilibrative nucleoside transporter (ENT1) promotes glutamate release by inhibition of adenosine signaling. However, whether ENT1 plays a role in epileptic seizure that involves elevated glutamatergic neurotransmission is unknown. Here, we report that both seizure rats and patients show increased expression of ENT1. Intrahippocampal injection of a specific inhibitor of ENT1, nitrobenzylthioinosine (NBTI), attenuates seizure severity and prolongs onset latency. In order to examine whether NBTI would be effective as antiepileptic after peripheral application, we injected NBTI intraperitoneally, and the results were similar to those obtained after intrahippocampal injection. NBTI administration leads to suppressed neuronal firing in seizure rats. In addition, increased mEPSC in seizure are inhibited by NBTI. Finally, NBTI results in deactivation of phosphorylated cAMP-response element-binding protein in the seizure rats. These results indicate that ENT1 plays an important role in the development of seizure. Inhibition of ENT1 might provide a novel therapeutic approach toward the control of epileptic seizure.

Human equilibrative nucleoside transporter 1 is not predictive for gemcitabine efficacy in advanced pancreatic cancer: translational results from the AIO-PK0104 phase III study with the clone SP120 rabbit antibody.

BACKGROUND: The role of human equilibrative nucleoside transporter 1 (hENT1) as a predictive biomarker for gemcitabine efficacy in advanced pancreatic cancer remains unclear to date. PATIENTS AND METHODS: AIO-PK0104 was a German multicenter phase III trial comparing gemcitabine/erlotinib followed by capecitabine (GEC) with capecitabine/erlotinib followed by gemcitabine (CEG) in advanced pancreatic cancer. Archival tumour tissue from 169 of the 274 eligible study patients was available for a central and standardised immunohistochemistry staining for hENT1 expression using the SP120 rabbit monoclonal anti-hENT1 antibody. Within a retrospective translational subgroup analysis, biomarker data were correlated with efficacy end-points. RESULTS: Thirty-nine out of 130 fresh-cut slides were scored as hENT1(high) (30%), whereas 91 samples were classified as hENT1(low) (70%). For the 62 patients randomised to CEG median overall survival was estimated with 6.4 months in the hENT1(low) compared to 6.9 months in the hENT1(high) subgroup (Hazard Ratio (HR) 0.88, 95% confidence interval (CI) 0.48-1.61, p=0.67). For the 68 patients randomised to GEC survival was 5.7 months in the hENT1(low) compared to 4.4 months in the hENT1(high) subgroup (HR 1.16, 95% CI 0.69-1.96, p=0.57). In 101 patients receiving gemcitabine at any time during study treatment (either within the 1st- or 2nd-line setting) hENT1(low) cases had a median overall survival of 7.5 months and hENT1(high) patients an overall survival of 4.4 months (HR 1.30, 95% CI 0.84-2.03, p=0.24), respectively. CONCLUSION: Within this subgroup analysis from Arbeitsgemeinschaft Internistische Onkologie-pancreatic cancer (AIO-PK0104), no evidence supporting the use of hENT1 as a predictive biomarker for gemcitabine efficacy in patients with advanced pancreatic cancer was found.

Gemcitabine-based chemoradiotherapy followed by surgery for borderline resectable and locally unresectable pancreatic ductal adenocarcinoma: significance of the CA19-9 reduction rate and intratumoral human equilibrative nucleoside transporter 1 expression.

OBJECTIVES: This study aimed to evaluate the efficacy of gemcitabine-based chemoradiotherapy followed by surgery (gem-CRTS) for pancreatic ductal adenocarcinoma (PDAC) for borderline resectable (BR) and locally unresectable (UR) tumors. METHODS: One hundred patients with PDAC who underwent the gem-CRTS protocol were classified into 3 groups, namely, resectable (R; 14), BR (44), and UR (42). After chemoradiotherapy, the patients were reassessed for curative-intent resection. RESULTS: At reassessment, distant metastases became apparent in 27% of R patients, in 12% of BR patients, and in 18% of UR patients. The multivariate analysis of preoperative factors indicated that the CA19-9 reduction rate was an independent prognostic factor in the BR group. Among reassessed patients, the resection rate was 63.6% in R, 83.7% in BR, and 50.0% in UR patients. In 63 patients that underwent curative-intent resection, the 3-year survival rate was 83.3% in R, 33.0% in BR, and 7.8% in UR patients. Using multivariate analysis, the independent prognostic factor was found to be the surgical margin in BR patients and human equilibrative nucleoside transporter 1 expression in UR patients. CONCLUSIONS: We consider that our gem-CRTS protocol, even for locally UR PDAC, allows for the identification of candidates for aggressive resection at the time of reassessment and improved prognosis in the patients with positive human equilibrative nucleoside transporter 1 expression.

Human equilibrative nucleoside transporter 1 (hENT1): do we really have a new predictive biomarker of chemotherapy outcome in pancreatic cancer patients?

Although systemic chemotherapy significantly improves the overall survival of pancreatic cancer patients, the prognosis remains extremely poor. The development of a drug resistance, either de novo or induced resistance, significantly limits the effectiveness of chemotherapy. SLC29A1 gene encodes human equilibrative nucleoside transporter 1 (hENT1) protein that is mediating the transport of nucleotides, both purines and pyrimidines, into the tumor cells. The aim of this mini-review is to summarize the current information concerning the prognostic and predictive role of SLC29A1 transporter (hENT1) expression in pancreatic cancer. Increased expression of SLC29A1 in vitro has been described as a potential critical factor determining the sensitivity of pancreatic cancer cells to gemcitabine and 5-fluorouracil, the principal cytotoxic agents used in the treatment of pancreatic cancer. The reports on the relationship between SLC29A1 expression and prognosis of patients with pancreatic cancer are currently rather conflicting. However, majority of studies on patients with resected pancreatic cancer have suggested that high SLC29A1expression may be predictive of improved survival in patients treated with gemcitabine. SLC29A1 has not been shown to represent a predictive biomarker for patients treated by 5-fluorouracil. In conclusion, potential prognostic and predictive role of SLC29A1 has been demonstrated for selected subset of patients.

Human equilibrative nucleoside transporter 1 (hENT1) predicts the Asian patient response to gemcitabine-based chemotherapy in pancreatic cancer.

BACKGROUND/AIMS: Individualized chemotherapy is important in the treatment of pancreatic cancer. Therefore, markers for predicting a patient response to treatment must be identified. We studied the relationship between human equilibrative nucleoside transporter 1 (hENT1) expression in tumor cells and the Asian patient response to gemcitabine-based chemotherapy. The aim of the study was to identify markers for individualized chemotherapy in Asian patients with pancreatic cancer. METHODOLOGY: Specimens from 44 Asian patients diagnosed with pancreatic adenocarcinoma were analyzed by immunohistochemistry for hENT1 expression in tumor cells. The correlations between hENT1 expression and various clinicopathological factors, including survival status, were studied. RESULTS: The overall survival (OS) and disease-free survival (DFS) in the hENT1 high-expression group were significantly longer than those of the hENT1 low or no-expression group: OS 21.75 months (95%CI=18.45-25.04 months) vs. 12.48 months (95%CI=10.12-14.85 months); DFS 15.44 months (95%CI=11.26-19.62 months) vs. 8.24 months (95%CI=8.69-9.78 months), respectively. CONCLUSIONS: Our studies suggest that hENT1 expression is related to the patient response to gemcitabine-based chemotherapy in Asian patients with pancreatic cancer. Therefore, hENT1 may be a valuable prognostic marker for individualized chemotherapy in pancreatic cancer.

Levels of gemcitabine transport and metabolism proteins predict survival times of patients treated with gemcitabine for pancreatic adenocarcinoma.

BACKGROUND & AIMS: Patients who undergo surgery for pancreatic ductal adenocarcinoma (PDAC) frequently receive adjuvant gemcitabine chemotherapy. Key determinants of gemcitabine cytotoxicity include the activities of the human equilibrative nucleoside transporter 1 (hENT1), deoxycytidine kinase (dCK), and ribonucleotide reductase subunit 1 (RRM1). We investigated whether tumor levels of these proteins were associated with efficacy of gemcitabine therapy following surgery. METHODS: Sequential samples of resected PDACs were retrospectively collected from 434 patients at 5 centers; 142 patients did not receive adjuvant treatment (33%), 243 received adjuvant gemcitabine-based regimens (56%), and 49 received nongemcitabine regimens (11%). We measured protein levels of hENT1, dCK, and RRM1 by semiquantitative immunohistochemistry with tissue microarrays and investigated their relationship with patients' overall survival time. RESULTS: The median overall survival time of patients was 32.0 months. Among patients who did not receive adjuvant treatment, levels of hENT1, RRM1, and dCK were not associated with survival time. Among patients who received gemcitabine, high levels of hENT1 and dCK were significantly associated with longer survival time (hazard ratios of 0.34 [P < .0001] and 0.57 [P = .012], respectively). Interaction tests for gemcitabine administration and hENT1 and dCK status were statistically significant (P = .0007 and P = .016, respectively). On multivariate analysis of this population, hENT1 and dCK retained independent predictive values, and those patients with high levels of each protein had the longest survival times following adjuvant therapy with gemcitabine. CONCLUSIONS: High levels of hENT1 and dCK in PDAC predict longer survival times in patients treated with adjuvant gemcitabine.

[The prognostic significance of human equilibrative nucleoside transporter1 (hENT1) expression in metastatic bladder cancer patients treated with gemcitabine-cisplatin based combination chemotherapy].

Systemic combination chemotherapy, such as the methotrexate, vinblastine, doxorubicin and cisplatin (MVAC) regimen, has shown certain activity in advanced bladder cancer, but is associated with a significant toxicity burden, with a treatment-related mortality of about 4%. Therefore, a great deal of interest has been focused on the gemcitabine-cisplatin (GC) combination chemotherapy which showed the same antitumor effect as MVAC chemotherapy with far less toxicity. Indeed, the GC regimen is now frequently administered as the first-line chemotherapy against metastatic bladder cancer. For the present, GC/MVAC regimens constitute alternative platform chemotherapy, until new evidence based strategy can be demonstrated. Accordingly it is important to be able to predict whether a regimen is effective in each patient with bladder cancer before the initiation of chemotherapy. Clinicopathological factors as the Karnofsky performance status and the presence of visceral metastases are well-established prognostic markers for poor survival. However, they are inadequate to predict the optimal therapeutic regimen for each individual patient. As for the predictive marker of cisplatin, ERCC1 may predict survival in bladder cancer treated by platinum-based therapy. The predictive potential of gemcitabine has not been previously considered in advanced bladder cancer treated by gemcitabine-combined systemic chemotherapy. In our retrospective study, the predictive value of a high expression level of hENT1 was assessed in bladder cancer treated by gemcitabine combined combination chemotherapy.

Disrupted plasma membrane localization of equilibrative nucleoside transporter 2 in the chemoresistance of human pancreatic cells to gemcitabine (dFdCyd).

Although the nucleoside pyrimidine analogue gemcitabine is the most effective single agent in the palliation of advanced pancreatic cancer, cellular resistance to gemcitabine treatment is a major problem in the clinical scene. To clarify the molecular mechanisms responsible for chemoresistance to gemcitabine, mRNA expression of the key enzymes including cytidine deaminase (CDA), deoxycytidine kinase (dCK), 5'-nucleotidase (NT5), equilibrative nucleoside transporter 1 and 2 (ENT1 and ENT2), dCMP deaminase (dCMPK), ribonucleotide reductase M1 and M2 (RRM1 and RRM2), thymidylate synthase (TS) and CTP synthase (CTPS) was examined. The interacellular uptake of gemcitabine was greatly impaired in the chemoresistant cell lines due to dysfunction of ENT1 and ENT2. Protein expression of ENT1 and ENT2 and their protein coding sequences were not altered. Immunohistochemical and western blot analyses revealed that localization of ENT2 on the plasma membrane was disrupted. These data suggest that the disrupted localization of ENT2 is one of causes of the impaired uptake of gemcitabine, resulting in a gain of chemoresistance to gemcitabine.

The differential expression of hCNT1 and hENT1 i n breast cancer and the possible impact on breast cancer therapy.

hCNT1 and hENT1, two members of the human nucleoside transporter families, expression levels were investigated, in normal and in breast tumour tissues, together with effects of gemcitabine cytotoxicity and in vivo tumour growth in MDA-MB-231 cells. hCNT1 and hENT1 levels were lower in tumour samples than in normal background tissue (p < 0.48). hENT1 levels decreased significantly with patient prognosis (disease free versus died from breast cancer, p = 0.047) although hCNT1 expression did not (disease free versus died from breast cancer, p = 0.97). Immunohistochemical staining of hCNT1 and hENT1 was stronger in normal than tumour tissue. hCNT1 knockdown caused MDA-MB-231 cells to be less sensitive to Gemcitabine compared with wild type and control plasmid cells (25% killed vs 88% and 90%). MDA MB-231 deltahENT1 (p = 0.139) and MDA MB-231deltahCNT1 (p = 0.033) tumours showed reduced growth compared with wild type, [71.99 +/- 39.81 mm3 MDA MB-231WT, 40.58 +/- 20.61 mm3 MDA MB-231 deltahCNT1 tumours, 51.58 +/- 49.29 mm3 MDA MB-231deltahENT1, 79.55 +/- 63.08 mm3 PEF and 57.92 +/- 21.67 mm3 GFP controls]. This study shows variability in hCNT1 and hENT1 expression in tumour and normal human breast tissue with different expression patterns related to patient prognosis and clinical outcome. The level of expression of CNT1 was closely linked to the cell's responsiveness to chemotherapeutic treatments.

Prognostic role of human equilibrative transporter 1 (hENT1) in patients with resected gastric cancer.

Nucleoside transporter proteins are specialized proteins that mediate the transport of nucleosides and nucleoside analog drugs across the plasma membrane. The human equilibrative nucleoside transporter 1 (hENT1) is a member of these proteins and mediates cellular entry of gemcitabine, cytarabine, and fludarabine. The hENT1 expression has been demonstrated to be related with prognosis and activity of gemcitabine-based therapy in breast, ampullary, lung, and pancreatic cancer. We investigated the immunohistochemical expression of hENT in tumor samples from 111 patients with resected gastric adenocarcinoma, correlating these data with clinical parameters and disease outcomes. None of the patients received chemotherapy or radiation therapy before or after surgery as a part of an adjuvant or neoadjuvant program. On univariate survival analysis, the hENT1 expression was associated with overall survival (OS) and disease free survival (DFS). Specifically, those patients with overexpression of hENT1 showed a shorter OS (P = 0.021) and a shorter DFS (P = 0.033). Considering only the node positive patients, higher hENT levels were associated with significantly shorter median DFS (21.7 months; 95% CI 11.1-32.4) compared with patients with low expression of hENT1. The hENT1 expression was defined, in the lymph-node positive patients, as an independent prognostic factor (P = 0.019). Furthermore, considering only patients with diffuse or mixed tumors and lymph-node positive, the expression of hENT1 was strongly related with DFS and OS. Immunohistochemistry for the hENT1 protein carries prognostic information in patients with resected gastric cancer and holds promise as a predictive factor in chemotherapy decisions.

Human equilibrative nucleoside transporter 1 levels predict response to gemcitabine in patients with pancreatic cancer.

BACKGROUND & AIMS: The human equilibrative nucleoside transporter (hENT1) protein transports gemcitabine into cells. Small retrospective studies in pancreatic cancer suggest that levels of hENT1 protein or messenger RNA may have prognostic value. We studied the predictive value of hENT1 levels in a cohort of pancreatic adenocarcinoma patients from the large prospective randomized adjuvant treatment trial RTOG9704. METHODS: In RTOG9704, 538 patients were assigned randomly, after surgical resection, to groups that were given either gemcitabine or 5-fluorouracil (5-FU). Immunohistochemistry for hENT1 was performed on a tissue microarray of 229 resected pancreatic tumors from RTOG9704 and scored as having no staining, low staining, or high staining. Associations between hENT1 protein and treatment outcome were analyzed by unconditional logistic regression analysis using the chi-square test and the Cox proportional hazards model. RESULTS: HENT1 expression was associated with overall and disease-free survival in a univariate (hazard ratio [HR], 0.51; 95% confidence interval [CI], 0.29-0.91; P= .02; and HR, 0.57; 95% CI, 0.32-1.00; P= .05) and multivariate model in the group given gemcitabine (HR, 0.40; 95% CI, 0.22-0.75; P= .004; and HR, 0.39; 95% CI, 0.21-0.73; P= .003). hENT1 expression was not associated with survival in the group given 5-FU. CONCLUSIONS: In this prospective randomized trial, hENT1 protein expression was associated with increased overall survival and disease-free survival in pancreatic cancer patients who received gemcitabine, but not in those who received 5-FU. These findings are supported by preclinical data; the gemcitabine transporter hENT1 is therefore a molecular and mechanistically relevant predictive marker of benefit from gemcitabine in patients with resected pancreatic cancer.

Human equilibrative nucleoside transporter 1 (hENT1) protein is associated with short survival in resected ampullary cancer.

BACKGROUND: Gemcitabine is an acceptable alternative to best supportive care in the treatment of advanced biliary tract cancers. The human equilibrative nucleoside transporter 1 (hENT1) is a ubiquitous protein and is the major means by which gemcitabine enters human cells. Moreover, recent reports indicate a significant correlation between immunohistochemical variations of hENT1 in tumor samples and survival after gemcitabine therapy in patients with solid tumors. MATERIALS AND METHODS: We used immunohistochemistry to assess the abundance and distribution of hENT1 in tumor samples from radically resected cancer of the ampulla, and sought correlations between immunohistochemical results and clinical parameters including disease outcomes. RESULTS: In the 41 individual tumors studied, 12 (29.3%) had uniformly high hENT1 immunostaining. Statistical analysis showed a significant correlation between hENT1 and Ki-67 (P = 0.04). No statistical significant differences were found between immunohistochemical findings and patient characteristics (sex, age, and tumor-node-metastasis). On univariate analysis, hENT1 and Ki-67 expression were associated with overall survival (OS). Specifically, those patients with overexpression of hENT1 showed a shorter OS (P = 0.022) and those with high Ki-67 staining showed a shorter survival (P = 0.05). CONCLUSIONS: hENT1 expression is a molecular prognostic marker for patients with resected ampullary cancer and holds promise as a predictive factor to assist in chemotherapy decisions.

Expression of human equilibrative nucleoside transporter 1 (hENT1) and its correlation with gemcitabine uptake and cytotoxicity in mantle cell lymphoma.

BACKGROUND AND OBJECTIVES: Nucleoside transporters might play a relevant role in the intracellular targeting of many nucleoside analogs used in anticancer therapy. Two gene families (SLC28 and SLC29) encode the two types of human nucleoside transporters, concentrative nucleoside transporter (CNT) and equilibrative nucleoside transporter (ENT) proteins. Chronic lymphocytic leukemia (CLL) cells express both SLC28- and SLC29-related mRNA, although transport function seems to be mostly related to ENT-type transporters. Here we have analyzed the role of nucleoside transporters in nucleoside-derived drug bioavailability and action in mantle cell lymphoma (MCL) cells. DESIGN AND METHODS: The relative amounts of hENT1 and hENT2-related mRNA and protein were analyzed in five MCL cell lines and 20 primary MCL tumors by real-time quantitative reverse transcriptase polymerase chain reaction and western blots. Cell viability, measured by annexin V-FITC staining, and nucleoside-derived drug transport were also studied. RESULTS: MCL cells express higher levels of hENT1 protein than do CLL cells, and a good correlation was found between protein and mRNA levels of hENT1, thus indirectly suggesting that hENT1 might be transcriptionally regulated in MCL cells. More importantly, a significant correlation between these two parameters, drug uptake and sensitivity to gemcitabine, was also observed. INTERPRETATION AND CONCLUSIONS: These results further support the concept that nucleoside transporters are implicated in the therapeutic response to nucleoside analogs, and suggest a particular and novel role for hENT1 in the genotoxic response to selected nucleoside analogs, such as gemcitabine, in MCL cells.