hENT1 Expression Research Articles

Human equilibrative nucleoside transporter-1 knockdown tunes cellular mechanics through epithelial-mesenchymal transition in pancreatic cancer cells.

We report cell mechanical changes in response to alteration of expression of the human equilibrative nucleoside transporter-1 (hENT1), a most abundant and widely distributed plasma membrane nucleoside transporter in human cells and/or tissues. Modulation of hENT1 expression level altered the stiffness of pancreatic cancer Capan-1 and Panc 03.27 cells, which was analyzed by atomic force microscopy (AFM) and correlated to microfluidic platform. The hENT1 knockdown induced reduction of cellular stiffness in both of cells up to 70%. In addition, cellular phenotypic changes such as cell morphology, migration, and expression level of epithelial-mesenchymal transition (EMT) markers were observed after hENT1 knockdown. Cells with suppressed hENT1 became elongated, migrated faster, and had reduced E-cadherin and elevated N-cadherin compared to parental cells which are consistent with epithelial-mesenchymal transition (EMT). Those cellular phenotypic changes closely correlated with changes in cellular stiffness. This study suggests that hENT1 expression level affects cellular phenotype and cell elastic behavior can be a physical biomarker for quantify hENT1 expression and detect phenotypic shift. Furthermore, cell mechanics can be a critical tool in detecting disease progression and response to therapy.

Abstract 918: Clinical validation of a multiplexed ChemoPlex SRM assay in FFPE human tumor tissue

Abstract Background: Current cancer treatment regimens rely on the use of chemotherapy agents that inhibit DNA replication and repair machinery. Several proteins are involved in this mechanism, such as TOPO1, TOPO2A, RRM1, FR-alpha and hENT1. The expression levels and activities of these proteins can greatly affect the success of chemotherapy; however current treatment indications are not based on tumor expression levels of these proteins. We have developed a quantitative, multiplexed ChemoPlex SRM method to evaluate these markers in a host of solid tumors from a limited amount of FFPE biopsy tissue using our Liquid Tissue®-SRM (LT-SRM) platform. Use of this method will enable a physician to understand individual tumor molecular machinery and ultimately could lead to individualized treatment decisions leading to better patient care. Methods: We used trypsin digestion mapping of recombinant proteins to identify optimal quantitative peptides for the ChemoPlex SRM assay. Standard curves were generated to determine the LOD, LOQ, accuracy, precision and linearity of the assay. The assay was pre-clinically validated on 14 cell lines with known expression levels of these Chemo-targets, and the assay was then run on microdissected archived FFPE human tissue samples from lung, gastro-esophageal cancer (GEC), breast, liver, colorectal, and ovarian tumors. Results: The peptides chosen for the 5 Chemo-Plex targets had LOD values of 150, 50, 300, 200, and 100 amol (CV<20%) for FR alpha, hENT1, TOPO1, TOPO2A, and RRM1, respectively. Fourteen cell lines were assayed for the Chemo-target expressions by LT-SRM, and regression analysis between protein and mRNA analysis for each target demonstrated varying correlations (R2=0.91(FR alpha); 0.78 (hENT1); 0.16 (TOPO1); 0.56 (TOPO2A); 0.59 (RRM1)) suggesting that RT-PCR measurements of mRNA levels would not be representative of cellular protein levels and therefore not useful for biomarker analysis for physicians. Our initial clinical analysis shows that FR alpha was detectable only in certain lung tumors (especially adenocarcinoma) and ovarian tumors; breast cancer tumors were found to have a wide range of hENT1 expression (LOD -1,284 amol/ug) while hENT1 expression in other tissues ranged from 90 to 377 amol/ug. TOPO1 had fairly ubiquitous expression (359 -1,300 amol/ug) except for one GEC and one breast cancer tissue. TOPO2A was identified in all tissue types (227-1,057 amol/ug). All samples except 1 GEC tissue express RRM1 (160 - 958 amol/ug) Discussion: We describe the development and initial clinical validation of a quantitative proteomic ChemoPlex SRM assay which accurately measures the expression of five chemotherapy targets in FFPE tumor tissue. When multiplexed along with other druggable biomarkers, the ChemoPlex SRM assay will allow more accurate identification of patients that are likely to benefit from the combination of chemotherapy and targeted therapies. Citation Format: Eunkyung An, Wei-Li Liao, Sheeno Thyparambil, Adele Blackler, Jamar Uzzell, Kathleen Bengali, Marlene Darfler, Jon Burrows, Todd Hembrough. Clinical validation of a multiplexed ChemoPlex SRM assay in FFPE human tumor tissue. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 918. doi:10.1158/1538-7445.AM2014-918

The hENT1 and DCK genes underlie the decitabine response in patients with myelodysplastic syndrome

Decitabine is approved for the treatment of MDS, but resistance to this agent is common. To determine the mechanisms underlying decitabine resistance, we measured the mRNA expression of metabolism (hENT1, DCK, CDA) and apoptosis (BCL2L10) genes and found that the hENT1 mRNA level was significantly higher in response compared with non-response patients (P=0.004). Furthermore, the DCK level was significantly reduced for relapse (P=0.012) compared with those with continued marrow CR (P=0.222). These findings indicate that the decitabine metabolic pathway affects its therapeutic effects, lower hENT1 expression may induce primary resistance and down-regulated DCK expression may be related to secondary resistance.

A phase II, open-label, multicenter study to evaluate the antitumor efficacy of CO-1.01 as second-line therapy for gemcitabine-refractory patients with stage IV pancreatic adenocarcinoma and negative tumor hENT1 expression

BackgroundNucleotide transporters such as human equilibrative nucleoside transporter-1 (hENT1) play a major role in transporting gemcitabine into cells. CO-1.01 (gemcitabine-5′-elaidate) is a novel cytotoxic agent consisting of a fatty acid derivative of gemcitabine, which is transported intracellularly independent of hENT1. CO-1.01 was postulated to have efficacy as a second-line treatment in gemcitabine-refractory pancreatic adenocarcinoma in patients with negative tumor hENT1 expression. MethodsEligibility criteria included patients with either a newly procured or archival biopsy tumor confirming the absence of hENT1 and either gemcitabine-refractory metastatic pancreas adenocarcinoma or with progression of disease following resection during or within 3 months of adjuvant gemcitabine therapy. Patients were treated with intravenous infusion of CO-1.01 dosed at 1250 mg/m2 on Days 1, 8, and 15 of a 4-week cycle. The primary end point was disease control rate (DCR). ResultsNineteen patients were enrolled of which 18 patients were evaluable for efficacy assessment. Thirteen patients (68%) had liver metastases, 6 (32%) had lymph node metastases, and 10 (53%) had lung metastases. Two of 18 patients (11%) achieved disease control. The median survival time was 4.3 (95% CI 2.1–8.1) months. All patients experienced at least one treatment-related adverse event with the majority of events being mild or moderate. ConclusionThis study did not meet its primary endpoint and no efficacy signal was identified for CO-1.01 in treating progressive metastatic pancreas adenocarcinoma.

Concurrent analysis of human equilibrative nucleoside transporter 1 and ribonucleotide reductase subunit 1 expression increases predictive value for prognosis in cholangiocarcinoma patients treated with adjuvant gemcitabine-based chemotherapy.

Background:The aim of this study was to investigate the predictive and prognostic values of intratumoural human equilibrative nucleoside transporter 1 (hENT1) and ribonucleotide reductase subunit 1 (RRM1) expression in advanced cholangiocarcinoma patients treated with adjuvant gemcitabine-based chemotherapy (AGC).Methods:Intratumoural hENT1 and RRM1 expression levels were investigated immunohistochemically in 127 patients with advanced cholangiocarcinoma who underwent surgical resection (68 with AGC and 59 without AGC). The impacts of hENT1 and RRM1 expression on survival were evaluated.Results:High intratumoural hENT1 and RRM1 expression levels were observed in 86 (68%) and 67 (53%) patients, respectively. In a multivariate analysis of 68 patients who received AGC, high hENT1 (P=0.044) and low RRM1 expression (P=0.009) were independently associated with prolonged disease-free survival (DFS), whereas low RRM1 expression (P=0.024) was independently associated with prolonged overall survival (OS). Moreover, concurrent high hENT1 and low RRM1 expression was a powerful independent predictor of prolonged DFS (P<0.001) and OS (P=0.001) when the combined classification of hENT1 and RRM1 was introduced.Conclusions:Concurrent analysis of hENT1 and RRM1 expression may increase the predictive value of these biomarkers for survival of advanced cholangiocarcinoma patients treated with AGC.

Gemcitabine and tegafur combination in adjuvant treatment of pancreatic cancer patients with low expression of hENT1

Gemcitabine and tegafur combination in adjuvant treatment of pancreatic cancer patients with low expression of hENT1

Results of up-front surgery combined with adjuvant chemotherapy for pancreatic head cancer: Adjuvant gemcitabine chemotherapy may be more effective for borderline resectable cases.

s / Pancreatology 14 (2014) S1eS129 S110 Conclusion: Clear trend in PFS increase in pancreatic cancer patients with low grade hENT1 expression treated with TG in adjuvant setting was revealed. Large-scale study is needed to prove our results and hypothesis.

Hent1 expression in patients with pancreatic cancer treated with gemcitabine after curative intended resection: Results from the CONKO-001 trial.

4124 Background: High expression of human equilibrative nucleosid transporter 1 (hent1) is considered to predict disease-free (DFS) and overall survival (OS) in patients (pts) treated with adjuvant...

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

BackgroundThe 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 methodsAIO-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. ResultsThirty-nine out of 130 fresh-cut slides were scored as hENT1high (30%), whereas 91 samples were classified as hENT1low (70%). For the 62 patients randomised to CEG median overall survival was estimated with 6.4months in the hENT1low compared to 6.9months in the hENT1high 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 5.7months in the hENT1low compared to 4.4months in the hENT1high 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) hENT1low cases had a median overall survival of 7.5months and hENT1high patients an overall survival of 4.4months (HR 1.30, 95% CI 0.84–2.03, p=0.24), respectively. ConclusionWithin 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.

Mo1574 Prognostic Impact of Human Equilibrative Nucleoside Transporter 1 and Ribonucleotid Reductase Submit 1 Expression in Cholangiocarcinoma Patients Treated With Adjuvant Gemcitabine-Based Chemotherapy

Background: The efficacy of AGC varies among individuals and the resulting survival rates are still unsatisfactory. Objective: The aim of this study was to investigate predictive value of intratumoral human equilibrative nucleoside transporter 1 (hENT1) and ribonucleotid reductase submit 1 (RRM1) expression for advanced cholangiocarcinoma patients treated with adjuvant gemcitabine-based chemotherapy (AGC) after surgical resection. Methods: Intratumoral hENT1 and RRM1 expression was investigated immunohistochemically in 127 patients with advanced cholangiocarcinoma who underwent surgical resection (including 68 with AGC, and 59 without AGC). Impact of hENT1 and RRM1 expression on diseasefree survival (DFS) and overall survival (OS) were evaluated by univariate and multivariate analyses. Results: High intratumoral hENT1 and RRM1 expression was observed in 86 (68%) and 67 (53%) patients, respectively. Univariate analysis of data from 68 patients with AGC revealed that high hENT1 and low RRM1 expression were significantly associated with longer DFS (hENT1, P = 0.005; RRM1, P = 0.015) and longer OS (hENT1, P = 0.036; RRM1, P = 0.035). In multivariate analysis, moderately or poorly differentiation, (P = 0.007), low hENT1expression (P = 0.044) and high RRM1 expression (P = 0.009) were identified as independent risk factors for poor DFS, and moderately or poorly differentiation, (P = 0.045), lymph node metastasis, (P = 0.011) and high RRM1 expression (P = 0.009) were identified as independent risk factors for poor OS. In contrast, hENT1 and RRM1 expression did not correlate with DFS and OS in 58 patients without AGC. Conclusions: Intratumoral hENT1 and RRM1 expression predicts survival of advanced cholangiocarcinoma patients treated with AGC.

Variability of Proliferation and Diffusion in Different Lung Cancer Models as Measured by 3'-Deoxy-3'-¹⁸F-Fluorothymidine PET and Diffusion-Weighted MR Imaging.

Molecular imaging allows the noninvasive assessment of cancer progression and response to therapy. The aim of this study was to investigate molecular and cellular determinants of 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) PET and diffusion-weighted (DW) MR imaging in lung carcinoma xenografts. Four lung cancer cell lines (A549, HTB56, EBC1, and H1975) were subcutaneously implanted in nude mice, and growth was followed by caliper measurements. Glucose uptake and tumor proliferation were determined by (18)F-FDG and (18)F-FLT PET, respectively. T2-weighted MR imaging was performed, and the apparent diffusion coefficient (ADC) was determined by DW MR imaging as an indicator of cell death. Imaging findings were correlated to histology with markers for tumor proliferation (Ki67, 5-bromo-2'-deoxyuridine [BrdU]) and cell death (caspase-3, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling). The expression of human equilibrative nucleoside transporter 1 (hENT1), thymidine kinase 1 (TK1), thymidylate synthase, and thymidine phosphorylase (TP) were analyzed by Western blot and immunohistochemistry. Thymidine levels were determined by liquid chromatography-mass spectrometry. Xenografts varied with respect to in vivo growth rates. MR imaging and PET revealed intratumoral heterogeneities, which were confirmed by histology. (18)F-FLT uptake differed significantly between tumor lines, with A549 and H1975 demonstrating the highest radiotracer accumulation (A549, 8.5 ± 3.2; HTB56, 4.4 ± 0.7; EBC1, 4.4 ± 1.2; and H1975, 12.1 ± 3.5 maximal percentage injected dose per milliliter). In contrast, differences in (18)F-FDG uptake were only marginal. No clear relationship between (18)F-FLT accumulation and immunohistochemical markers for tumor proliferation (Ki67, BrdU) as well as hENT1, TK1, or TS expression was detected. However, TP was highly expressed in A549 and H1975 xenografts, which was accompanied by low tumor thymidine concentrations, suggesting that tumor thymidine levels influence (18)F-FLT uptake in the tumor models investigated. MR imaging revealed higher ADC values within proliferative regions of H1975 and A549 tumors than in HTB56 and EBC1. These ADC values were negatively correlated with cell density but not directly related to cell death. A direct relationship of (18)F-FLT with proliferation or ADC with cell death might be complicated by the interplay of multiple processes at the cellular and physiologic levels in untreated tumors. This issue must be considered when using these imaging modalities in preclinical or clinical settings.

Erythrocyte membrane proteins in sickle cell disease (598.1)

Sickle cell disease (SCD) is a common inherited hematological disorder where mutation causes substitution of valine for glutamate in β‐globin, giving hemoglobin S (HbS). HbS polymerization and cell dehydration cause cell distortion (sickling) at low oxygen tension, often leading to microvascular occlusion. Membrane changes are found in SCD, and increased Na+/H+ exchange activity mediated by NHE‐1 transporter has been reported. This study assayed levels of key nutrient (GLUT‐1 and hENT‐1) and ion (AE‐1 and NHE‐1) transporters to better understand pathophysiological changes in SCD.Membrane protein levels were examined by SDS‐PAGE and quantitative immunoblot analysis (against a common standard arbitrarily set as 100%). Sodium pump (Na/K ATPase) activity was followed by surrogate assay of p‐nitrophenylphosphatase (PNPase) activity.HPLC analysis confirmed HbSS and control phenotypes. SCD showed decreased expression of hENT‐1 (75.33 ± 3.75 n=11 vs. 98.09 ± 7.67 n=11; p &lt; 0.05), elevation of NHE‐1 (108.92 ± 5.01 vs. 93.19 ± 4.90; p &lt; 0.05) and GLUT‐1 (116.92 ± 5.45 vs. 90.94 ± 2.55; p &lt; 0.05) but no significant change in AE‐1 (96.89 ± 2.99 vs. 95.55 ± 2.48) between normal and SCD samples. PNPase activity was increased in SCD (1.303 ± 0. 211 vs. 0.729 ± 0.137 µmole/mg/ml, n= 7; p &lt; 0.05).NHE‐1 increase is consistent with reported Na‐H exchange and decreased hENT‐1 may affect plasma and intracellular purine levels in SCD.Grant Funding Source: Supported by the College of Graduate Studies, Kuwait University, Research Sector Kuwait University R

Human Equilibrative Nucleoside Transporter 1 Predicts Survival in Patients with Pancreatic Cancer Treated with Gemcitabine: A Meta-Analysis

Increasing scientific evidence suggests that human equilibrative nucleoside transporter 1 (hENT1) may be a powerful predictor of survival in patients with pancreatic cancer treated with adjuvant gemcitabine-based chemotherapy after operative resection, but many existing studies have yielded inconclusive results. This meta-analysis aims to assess the prognostic role of hENT1 in predicting survival in patients with pancreatic cancer treated with gemcitabine. An extensive literature search for relevant studies was conducted on PubMed, Embase, Web of Science, Cochrane Library, and CBM databases from their inception through May 1, 2013. This meta-analysis was performed using the STATA 12.0 software. The crude hazard ratio (HR) with 95% confidence interval (CI) was calculated. Eleven clinical studies were included in this meta-analysis with a total of 851 pancreatic cancer patients, including 478 patients in the high hENT1 expression group and 373 patients in the low hENT1 expression group. Our meta-analysis revealed that high hENT1 expression was associated with improved overall survival (OS) of pancreatic cancer patients (HR=2.61, 95% CI=2.02-3.34). Pancreatic cancer patients with high hENT1 expression also had a longer disease-free survival (DFS) than those with low hENT1 expression (HR=2.62, 95% CI=1.94-3.54). Further, high hENT1 mRNA showed significant association with improved OS and DFS of pancreatic cancer patients (HR=2.65, 95% CI=1.75-4.00; HR=3.29, 95% CI=1.85-5.84; respectively). In conclusion, our meta-analysis suggests that high hENT1 expression may be associated with improved OS and DFS of pancreatic cancer patients treated with gemcitabine. Detection of hENT1 expression may be a promising biomarker for gemcitabine response and prognosis in pancreatic cancer patients.

A phase II study of elacytarabine in combination with idarubicin and of human equilibrative nucleoside transporter 1 expression in patients with acute myeloid leukemia and persistent blasts after the first induction course

Unlike cytarabine, cellular entry of Elacytarabine, the elaidic acid ester derivative of cytarabine, is independent of the human equilibrative nucleoside transporter 1 (hENT1). This phase II study tested whether the hENT1 blast expression level can be used as a predictive marker for cytarabine response and if the efficacy of elacytarabine is independent of hENT1 expression. A total of 51 patients with acute myeloid leukemia (AML) induction failure were given elacytarabine–idarubicin as a second induction course. The hENT1 expression level was analyzed prior to first induction and/or prior to treatment with elacytarabine. The overall response rate (ORR) was 41% and the safety profile was manageable. There is a trend suggesting that hENT1 expression influences response to cytarabine, but not sufficient to support it as a biomarker for guiding treatment. Further, we conclude that the activity of elacytarabine is not significantly predicted by the hENT1 expression level.

Human equilibrative nucleoside transporter 1 (hENT1) in gemcitabine and FOLFOX (oxaliplatin, 5-fluorouracil, and leucovorin)-treated patients with metastatic pancreatic cancer: The randomized phase II PAN1 study.

228 Background: GEM-based treatment is a standard treatment for metastatic pancreas cancer. Monotherapy efficacy is only modest, and outcomes are improved by combination treatment or the non-GEM FOLFIRINOX triplet. There is a need for biomarkers to select between these divergent options. hENT1 is a potential predictive marker of benefit from GEM, but conflicting results have been observed in studies that have evaluated resected and metastatic pancreatic cancer. This principal objective of this study was to evaluate hENT1 prospectively as a predictive marker in GEM and non-GEM treated patients. Methods: Patients with previously untreated metastatic pancreatic adenocarcinoma and tumour tissue available for hENT1 testing were randomized between GEM (1,000 mg/m2 D1, 8, 15 Q4w) or FOLFOX (oxaliplatin 85 mg/m2, 5-fluorouracil (5FU) 400 mg/m2, leucovorin 400 mg/m2 D1 and 5FU 2,400 mg/m2over 46 hours D1-3) until disease progression or unacceptable toxicity. Tumor samples were tested prospectively for hENT1 by immunohistochemistry. The primary endpoint was progression-free survival (PFS). Results: The study was open from July 2011 to February 2013, but closed after 16 of the planned 80 patients were enrolled. 1 patient who died before treatment was excluded from the outcome analysis. 7/16 patients were hENT1 high (43.8%, 95%CI 23.1-66.8%). In the GEM arm (n=7), the 4 who were hENT1 high had longer PFS and overall survivals (OS) than those who were hENT1 low (median PFS 5.7 vs. 1.8 months; median OS 13.9 vs. 3.5 months), whereas outcomes were similar with FOLFOX for both hENT1 groups. There were no unexpected adverse events. Conclusions: This prospective study showed a longer survival in those patients with high hENT1 treated with GEM. There was no difference in survival according to hENT1 expression in patients treated with FOLFOX. Given the small sample size this may be due to chance, but supports the need for further research into the predictive value of hENT1. Clinical trial information: ACTRN12610001047088.[Table: see text]

HENT1 expression is predictive of gemcitabine outcome in pancreatic cancer: a systematic review.

High human equilibrative nucleoside transporter 1 (hENT1)-expression has shown a survival benefit in pancreatic cancer patients treated with gemcitabine in several studies. The aim of this systematic review was to summarize the results and try to assess the predictive value of hENT1 for determining gemcitabine outcome in pancreatic cancer. Relevant articles were obtained from PubMed, Embase and Cochrane databases. Studies evaluating hENT1-expression in pancreatic tumor cells from patients treated with gemcitabine were selected. Outcome measures were overall survival, disease-free survival (DFS), toxicity and response rate. The database searches identified 10 studies that met the eligibility criteria, and a total of 855 patients were included. Nine of 10 studies showed a statistically significant longer overall survival in univariate analyses in patients with high hENT1-expression compared to those with low expression. In the 7 studies that reported DFS as an outcome measure, 6 had statistically longer DFS in the high hENT1 groups. Both toxicity and response rate were reported in only 2 articles and it was therefore hard to draw any major conclusions. This review provides evidence that hENT1 is a predictive marker for pancreatic cancer patients treated with gemcitabine. Some limitations of the review have to be taken into consideration, the majority of the included studies had a retrospective design, and there was no standardized scoring protocol for hENT1-expression.

Identification of Primary Equilibrative Nucleoside Transporter 1 mRNA Isoforms Resulting from Alternative Promoter Usage in Human Hepatocytes

Human equilibrative nucleoside transporter 1 (hENT1) transports various nucleoside analogues into cells. Although the single hENT1 promoter region (P1) and the mRNA isoform (a1) have been characterized previously, we have recently identified additional promoter regions P2 and P3 (which primarily generate c1/2/3 mRNAs and d1/2/3/4 mRNAs, respectively) in the human liver. Therefore, this study aimed at identifying the primary hENT1 mRNA isoforms expressed in human hepatocytes, while simultaneously obtaining functional evidence of alternative hENT1 promoter usage. Our results showed that the expressions of hENT1c1, d3, and (to a lesser extent) c2 mRNAs were strikingly predominant over the other mRNA isoforms in human hepatocytes, that the abundant expression of these mRNAs was consistent with the high levels of P2 and P3 promoter activity, and that these promoters were significantly marked by transcriptionally active histone modification in hepatic cells. To summarize, our results demonstrate that, resulting from the manipulated alternative promoter usage, hENT1c1 and d3 (and c2) mRNAs are primarily expressed in human hepatocytes, which suggests that they may play important roles in controlling hENT1 expression levels in those cells. Our findings are expected to provide significant insights into the molecular machinery of hENT1 expression control.

Pancreatic Cancer hENT1 Expression and Survival From Gemcitabine in Patients From the ESPAC-3 Trial

Human equilibrative nucleoside transporter 1 (hENT1) levels in pancreatic adenocarcinoma may predict survival in patients who receive adjuvant gemcitabine after resection. Microarrays from 434 patients randomized to chemotherapy in the ESPAC-3 trial (plus controls from ESPAC-1/3) were stained with the 10D7G2 anti-hENT1 antibody. Patients were classified as having high hENT1 expression if the mean H score for their cores was above the overall median H score (48). High and low hENT1-expressing groups were compared using Kaplan-Meier curves, log-rank tests, and Cox proportional hazards models. All statistical tests were two-sided. Three hundred eighty patients (87.6%) and 1808 cores were suitable and included in the final analysis. Median overall survival for gemcitabine-treated patients (n = 176) was 23.4 (95% confidence interval [CI] = 18.3 to 26.0) months vs 23.5 (95% CI = 19.8 to 27.3) months for 176 patients treated with 5-fluorouracil/folinic acid (χ(2) 1=0.24; P = .62). Median survival for patients treated with gemcitabine was 17.1 (95% CI = 14.3 to 23.8) months for those with low hENT1 expression vs 26.2 (95% CI = 21.2 to 31.4) months for those with high hENT1 expression (χ(2)₁= 9.87; P = .002). For the 5-fluorouracil group, median survival was 25.6 (95% CI = 20.1 to 27.9) and 21.9 (95% CI = 16.0 to 28.3) months for those with low and high hENT1 expression, respectively (χ(2)₁ = 0.83; P = .36). hENT1 levels were not predictive of survival for the 28 patients of the observation group (χ(2)₁ = 0.37; P = .54). Multivariable analysis confirmed hENT1 expression as a predictive marker in gemcitabine-treated (Wald χ(2) = 9.16; P = .003) but not 5-fluorouracil-treated (Wald χ(2) = 1.22; P = .27) patients. Subject to prospective validation, gemcitabine should not be used for patients with low tumor hENT1 expression.

Assessment Of Human Equilibrative Nucleoside Transporter 1 (hENT1) – Expression By Flow Cytometry In Acute Myeloid Leukemia and Myelodysplastic Syndromes and Correlation To Clinical Outcome In Acute Myeloid Leukemia Patients Treated With Intensive Chemotherapy

Assessment Of Human Equilibrative Nucleoside Transporter 1 (hENT1) – Expression By Flow Cytometry In Acute Myeloid Leukemia and Myelodysplastic Syndromes and Correlation To Clinical Outcome In Acute Myeloid Leukemia Patients Treated With Intensive Chemotherapy

FLT3 Is Involved In Ara-C Transport By Human Equilibrative Nucleoside Transporter (hENT1) In Pediatric Acute Leukemia

IntroductionDespite the remarkable improvement in survival of children with acute leukemia in the last decades, some patients still have a poor outcome. FMS-like tyrosine Kinase 3 (FLT3) is a tyrosine-kinase receptor with a key role in hematopoiesis whose mutations and overexpression have emerged as negative prognostic biomarkers in childhood leukemia. Infant leukemias (those diagnosed at age<1 year) are a special subgroup characterized by distinctive clinico-biological features, including frequent MLL (Mixed Lineage Leukemia) gene rearrangements, high FLT3 expression and high sensitivity to cytarabine (Ara-C), but a dismal prognosis. Ara-C is a deoxycytidine analog known to be transported by the human equilibrative nucleoside transporter 1 (hENT1) across the cell membrane. Once inside the cell Ara-C is activated by deoxycitidine kinase (DCK) to finally exert its cytotoxic action. High hENT1 expression levels have been involved in Ara-C sensitivity in patients with acute leukemia. However, the mechanisms that regulate the expression of hENT1 and its activity, as well as the putative relationship between FLT3 and Ara-C transport and metabolism are scarcely known. AimTo study the role of FLT3 in the regulation of the expression and activity of the main Ara-C transporters and metabolizing enzymes (ME) in pediatric leukemia. Patients, Materials, and MethodsBone marrow samples of 56 pediatric patients diagnosed with acute leukemia in Hospital Sant Joan de Déu and 3 acute leukemia cell lines (MV4-11, SEM, K562) were used for screening of Ara-C transporters and ME expression. In all cases the patients or their parents signed an informed consent. We performed a positive selection of cases with acute lymphoblastic leukemia (ALL) with hyperdiploidy (>50 chromosomes) and of MLL-rearranged cases presenting either as ALL or acute myeloblastic leukemia (AML), as these are the subgroups with highest expression of FLT3 reported in the literature, although other leukemia subtypes were also represented. The mRNA expression levels of FLT3, nucleoside transporters (NT) (hENT1, hENT2, hCNT1, hCNT3) and Ara-C ME, dCK and CNII, were quantified using real-time PCR and analyzed with the 2-ΔΔCt method, with non-neoplastic samples used as controls for the relative quantification. Direct nucleoside and Ara-C uptake measurements were performed using [5,6-3H]-nucleosides. The role FLT3 might play in the expression of NT and ME genes as well as on the activity of Ara-C transporters in these cell lines was addressed by repressing FLT3 function with its specific inhibitor PKC412 (sold only for research purposes). ResultsWe included 56 patients (68% males) diagnosed with acute leukemia. The median age was 5.3 years (range 0-16.2), with 3 cases of infant leukemia. Fifty cases (89%) were precursor B-ALL (24 hyperdiploid cases, 5 MLL rearranged, 3 BCR-ABL+, 4 E2A-PBX1+, 5 TEL-AML1+, 9 other subtypes), 5 cases were AML (4 MLL positive cases and one case with mutated FLT3) and one case was a T-ALL harboring FLT3 mutation. As expected, the FLT3 expression was higher in cases with ALL and MLL rearrangement and, to a less extent, in ALL with hyperdiploidy. Interestingly, a significant positive correlation was found between FLT3 and hENT1 expression (mRNA levels) with all patient samples (figure 1). hENT1 expression and cytarabine-mediated uptake was significantly repressed when MV4-11 and SEM cell lines were exposed to the FLT3 inhibitor PKC412 (figures 2 & 3) without affecting hENT2, CNT1 and CNT3 expression and activity. [Display omitted] [Display omitted] [Display omitted] ConclusionsOur results show a strong correlation between FLT3 and the Ara-C transporter hENT1 in pediatric leukemia patients. This observation was consistent with the in vitro evidence that FLT3 inhibition resulted in hENT1 repression and down-regulation of Ara-C uptake in leukemic derived cell lines. Taken together, our data suggest that FLT3 regulates hENT1, thereby modulating the ability of cancer cells to incorporate Ara-C and promote its cytotoxic action. As FLT3 inhibitors are currently being tested as mono or combined therapy with Ara-C in several clinical trials, based upon our observations we suggest that a better schedule design might eventually be needed when dealing with treatments involving FLT3 inhibitors and Ara-C, thereby improving the outcome of this subset of patients. Disclosures:Off Label Use: The presentation include the results of in vitro studies with the FLT3 inhibitor PKC412. This drug was only used for in vitro studies, exclusively for research purposes.