High hENT1 Expression Research Articles

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

BackgroundFOLFIRINOX 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 β-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. MethodsWe 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. ResultsIn 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. ConclusionOur 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.

HENT1’s role in adjuvant intra-arterial gemcitabine-based chemotherapy for resectable pancreatic cancer patients

BackgroundWe aimed to verify the role of hENT1 as a prognostic predictor for patients with resectable pancreatic ductal adenocarcinoma (PDAC) who underwent radical resection followed by intra-arterial infusion of gemcitabine-based regimen.MethodsWe collected surgical samples from 102 patients with resectable PDAC who received radical resection followed by intra-arterial infusion of gemcitabine-based regimen. The hENT1 expression with the help of immunohistochemistry was conducted using formalin-fixed and paraffin embedded tissues. The Kaplan–Meier analyses and Cox regression were used to evaluate the mortality hazard associated with the discrepancy between strong and weak of hENT1 expression. Patients’ clinical and pathological characteristics were compared between the two groups, then the role of hENT1 as a prognostic predictor was further explored.ResultsA total of 102 patients were included to assess the hENT1 expression. 50 patients were classified into high hENT1 expression group, the other 52 patients were attributed into low hENT1 expression group. High hENT1 expression was related to a significantly improved overall survival (OS) (p = 0.014) and disease-free survival (DFS) (p = 0.004). Both univariate (p = 0.001) and multivariate analyses (p < 0.001) indicated that high hENT1 expression was related to a decreased mortality.ConclusionsHigh expression of hENT1 is positive prognostic factor for adjuvant intra-arterial gemcitabine-based chemotherapy in resectable PDAC.

Human equilibrative nucleoside transporter 1 (hENT1) and deoxycytidine kinase (DCK) are predictive biomarkers for Gemcitabine-based chemoradiotherapy in resected pancreatic cancer patients: a retrospective cohort study

Background and Aim Pancreatic cancer is reported to be one of the cancer-related deaths all over the world. Gemcitabine has been identified as one of the important therapies of malignant diseases. Gemcitabine cellular uptake is allowed by the transporter protein, human equilibrative nucleoside transporter 1 (hENT1). Gemcitabine is phosphorylated by deoxycytidine kinase (dCK) to its active metabolites intracellularly. This study aimed to determine retrospectively the correlation between hENT1 and dCK expression by immunohistochemistry and the overall survival outcome in pancreatic cancer patients who underwent resection and received gemcitabine chemotherapy postoperatively. Materials and methods The immunohistochemical analysis was accomplished on samples from 140 patients, who underwent pancreatic cancer resection and was treated by gemcitabine. Immunostaining was done by two expert pathologists and both are blind to patient’s characteristics. The intensity of staining of the tumor determines the scoring for hENT1 and dCK. Results Of the 140 tissue samples, 73 (52.1%) had detectable hENT1 cell membrane immunostaining and 67 samples (47.8%) without detectable hENT1. Patients who showed no significant hENT1 immunostaining had a median survival of 6 months, while patients who showed significant hENT1 staining had a median survival longer by 17 months. Of the 140 tissue samples, 52 (37.1%) had detectable dCK cytoplasmic immunostaining and 88 samples (62.9%) without detectable dCK. Patients who showed no significant dCK immunostaining had a median survival of 6.57 months, while patients who showed significant dCK staining had a median survival longer by 19 months. Conclusion In patients who received gemcitabine after pancreatic resection, a high expression of hENT1 and dCK are significantly correlated with a longer survival. In addition, in patients who underwent pancreatic cancer resection, immunohistochemical analysis of hENT1 and dCK are considered significant predictive markers to choose patients responding to gemcitabine therapy.

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.

BackgroundExpression 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.AimWe aimed to investigate the concordance of immunohistochemical hENT1 expression between the two antibodies and prognosis.MethodsThe 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.ResultsThe 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.ConclusionThe 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.

Human equilibrative nucleoside transporter-1 (hENT1) and ribonucleotide reductase regulatory subunit M1 (RRM1) expression; do they have survival impact to pancreatic cancer?

Backgrounds/AimsGemcitabine is still one of adjuvant options in chemotherapeutic agent for pancreatic ductal adenocarcinoma (PDAC). Integral membrane transporter protein and intracellular enzymes including human equilibrative nucleoside transporter 1 (hENT1), deoxycytidine kinase (dCK), ribonucleotide reductase (RR) M1, and M2 are known as important factors for chemosensitivity of gemcitabine. We aimed to investigate the correlation between these key molecules and 5-year actual survival in PDAC patients.MethodsThe expression of intratumoral hENT1, dCK, RRM1, and RRM2 was assessed immunohistochemically in 160 PDAC patients underwent surgical resection. Association between clininopathologic factors, immunohistochemical results, and overall survival were analyzed.ResultsAdjuvant chemotherapy including concurrent chemoradiotherapy was not associated with overall survival (HR, 0.92; 95% CI, 0.65-1.31; p=0.658). High hENT1 expression group did not show statistical survival difference, compared with all others (HR, 1.16; 95% CI, 0.82-1.65, p=0.396). Gemcitabine therapy and high hENT1 group was compared with all other patients, and no difference in overall survival was identified (HR, 0.99; 95% CI, 0.68-1.42; p=0.940). And, gemcitabine therapy and high hENT1 group did not differ statistically from gemcitabine therapy and low hENT1 expression (HR, 0.92; 95% CI, 0.55-1.56; p=0.764). The intensity of dCK, RRM1, and RRM2 expression was not associated with overall survival (p=0.413, p=0.138 and p=0.061) in univariate analysis.ConclusionsThe expression of hENT1, dCK, RRM1 and RRM2 may not be associated with overall survival for patients with pancreatic cancer on gemcitabine adjuvant therapy. These proteins and other factors that may interact with or confound these results should be investigated in the near future.

Human equilibrative nucleoside transporter-1 expression is a predictor in patients with resected pancreatic cancer treated with adjuvant S-1 chemotherapy.

The high expression of human equilibrative nucleoside transporter‐1 (hENT1) and the low expression of dihydropyrimidine dehydrogenase (DPD) are reported to predict a favorable prognosis in patients treated with gemcitabine (GEM) and 5‐fluorouracil (5FU) as the adjuvant setting, respectively. The expression of hENT1 and DPD were analyzed in patients registered in the JASPAC 01 trial, which showed a better survival of S‐1 over GEM as adjuvant chemotherapy after resection for pancreatic cancer, and their possible roles for predicting treatment outcomes and selecting a chemotherapeutic agent were investigated. Intensity of hENT1 and DPD expression was categorized into no, weak, moderate or strong by immunohistochemistry staining, and the patients were classified into high (strong/moderate) and low (no/weak) groups. Specimens were available for 326 of 377 (86.5%) patients. High expression of hENT1 and DPD was detected in 100 (30.7%) and 63 (19.3%) of 326 patients, respectively. In the S‐1 arm, the median overall survival (OS) with low hENT1, 58.0 months, was significantly better than that with high hENT1, 30.9 months (hazard ratio 1.75, P = 0.007). In contrast, there were no significant differences in OS between DPD low and high groups in the S‐1 arm and neither the expression levels of hENT1 nor DPD revealed a relationship with treatment outcomes in the GEM arm. The present study did not show that the DPD and hENT1 are useful biomarkers for choosing S‐1 or GEM as adjuvant chemotherapy. However, hENT1 expression is a significant prognostic factor for survival in the S‐1 arm.

Predictive and Prognostic Properties of Human Equilibrative Nucleoside Transporter 1 Expression in Gemcitabine-Treated Pancreatobiliary Cancer: A Meta-Analysis.

Gemcitabine, the primary drug for the treatment of pancreatobiliary cancer (PBC), requires human equilibrative nucleoside transporter 1 (hENT1) to enter cells. High tumoral hENT1 expression has been linked with improved survival among patients with PBC treated with gemcitabine; however, this finding has been inconsistent, and studies used different expression assays. Databases were reviewed for studies that examined hENT1 and clinical outcome in PBC. Of 307 publications, 34 studies were found that used immunohistochemistry (IHC) with one of eight anti-hENT1 antibody assays. Five studies were excluded for redundancy, and 29 studies underwent detailed review. On average, 51% of tumor samples had high hENT1 expression (range, 7% to 92%). Among studies that examined hENT1 expression and overall survival (OS), 58% (15 of 26 studies) showed an association between high tumoral hENT1 and improved OS for gemcitabine-treated patients. Among 10D7G2 antibody studies, 88% (seven of eight studies) demonstrated this association. Studies with other antibodies-in particular, SP120 (two of nine studies)-were less consistent. The ability to detect an association between improved OS and high hENT1 was antibody dependent (χ2 P = .0237). An association between high tumoral hENT1 expression and improved disease-free/progression-free survival (DFS/PFS) was demonstrated in 71% of studies (15 of 21 studies). Pooled hazard ratio (HR) analyses of all antibody studies demonstrated a link between high hENT1 tumor expression and improved OS (HR, 0.674; 95% CI, 0.509 to 0.893; P = .006) and DFS/PFS (HR, 0.740; 95% CI, 0.517 to 0.1.059; P = .10). This signal was stronger among studies that used the 10D7G2 antibody in comparison to those in which another antibody was used, with HRs of 0.488 (95% CI, 0.396 to 0.602; P < .001) and 0.410 (95% CI, 0.280 to 0.599; P < .001), respectively. High tumoral hENT1 expression on IHC with 10D7G2 is a strong and reproducible prognostic marker for improved outcome among gemcitabine-treated patients with PBC.

Effect of hypoxia on human equilibrative nucleoside transporters hENT1 and hENT2 in breast cancer.

Elevated proliferation rates in cancer can be visualized with positron emission tomography (PET) using 3'-deoxy-3'-l-[18F]fluorothymidine ([18F]FLT). This study investigates whether [18F]FLT transport proteins are regulated through hypoxia. Expression and function of human equilibrative nucleoside transporter (hENT)-1, hENT2, and thymidine kinase 1 (TK1) were studied under normoxic and hypoxic conditions, and assessed with [18F]FLT-PET in estrogen receptor positive (ER+)-MCF7, triple-negative MDA-MB231 breast cancer (BC) cells, and MCF10A cells (human mammary epithelial cells). Functional involvement of hENT2 [18F]FLT transport was demonstrated in all cell lines. In vitro [18F]FLT uptake was higher in MDA-MB231 than in MCF7: 242 ± 9 vs. 147 ± 18% radioactivity/mg protein after 60 min under normoxia. Hypoxia showed no significant change in radiotracer uptake. Protein analysis revealed increased hENT1 (P < 0.0963) in MDA-MB231. Hypoxia did not change expression of either hENT1, hENT2, or TK1. In vitro inhibition experiments suggested involvement of hENT1, hENT2, and human concentrative nucleoside transporters during [18F]FLT uptake into all cell lines. In vivo PET imaging revealed comparable tumor uptake in MCF7 and MDA-MB231 tumors over 60 min, reaching standardized uptake values of 0.96 ± 0.05 vs. 0.89 ± 0.08 (n = 3). Higher hENT1 expression in MDA-MB231 seems to drive nucleoside transport, whereas TK1 expression in MCF7 seems responsible for comparable [18F]FLT retention in ER+ tumors. Our study demonstrates that hypoxia does not significantly affect nucleoside transport as tested with [18F]FLT in BC.-Krys, D., Hamann, I., Wuest, M., Wuest, F. Effect of hypoxia on human equilibrative nucleoside transporters hENT1 and hENT2 in breast cancer.

Prognostic immunohistochemical biomarkers of chemotherapy efficacy in biliary tract cancer: A systematic review and meta-analysis.

Chemotherapy is the mainstay of systemic treatment of biliary tract cancer (BTC). However, the treatment response to chemotherapy varies between patients. Currently, no prognostic biomarkers for chemotherapy efficacy have been considered for use in clinical practice. A systematic review was conducted to evaluate the prognostic value of immunohistochemical biomarkers for chemotherapy in patients with resected as well as with advanced BTC. Medline and EMBASE databases were searched up to March 2017 for studies that evaluated biomarker expression by immunohistochemistry in resected or advanced BTC patients treated with chemotherapy. The primary endpoints were overall survival (OS) and disease or progression free survival (DFS or PFS). Twenty-six studies, including a total of 1348 patients and 26 different biomarkers, met the inclusion criteria and were included in this review. The most frequently studied prognostic biomarkers in BTC were the human Equilibrative Nucleoside Transporter 1 (hENT1), Ribonucleotide Reductase M1 (RRM1), and excision repair cross-complementation 1 (ERCC1). In the meta-analysis of patients treated with gemcitabine-based chemotherapy, high hENT1 expression was associated with longer OS (HR 0.43, 95% CI: 0.28 to 0.64) and DFS/PFS (HR 0.45, 95% CI: 0.33 to 0.61). hENT1 is a promising prognostic biomarker for gemcitabine-based chemotherapy in resected as well as in advanced BTC and should be further validated for the selection of patients for chemotherapy.

Human equilibrative nucleoside transporter 1 gene expression is associated with gemcitabine efficacy in advanced leiomyosarcoma and angiosarcoma

Background:The expression of human equilibrative nucleoside transporter 1 (hENT1), the major gemcitabine transporter into cells, has been thoroughly investigated as a predictive marker of response to gemcitabine in pancreatic cancer and biliary tract cancers. Since gemcitabine is widely used in the treatment of leiomyosarcoma and angiosarcoma, we investigated the correlation between hENT1 expression and gemcitabine efficacy in these sarcoma subtypes.Methods:We retrospectively identified 71 patients affected by advanced angiosarcoma (26) or leiomyosarcoma (45) treated within five Italian referral centres for sarcoma; among them, 49 patients (15 angiosarcoma, 34 leiomyosarcoma) were treated with gemcitabine. All tumour samples were analysed for hENT1 expression by real-time PCR. Median 2–ΔCt value was used as the cutoff to dichotomise patients into ‘high’ expression and ‘low’ expression groups. Kaplan–Meier analysis was performed to estimate progression-free survival (PFS) and overall survival (OS).Results:We found a significant association between high hENT1 expression levels and favourable outcome in terms of PFS and OS compared to cases with low hENT1 expression in leiomyosarcoma treated with gemcitabine (PFS: 6.8 vs 3.2 months, P=0.004; OS: 14.9 vs 8.5 months, P=0.007). In addition, hENT1 overexpression correlated with a significant improvement in PFS (9.3 vs 4.5 months; P=0.02) and OS (20.6 vs 10.8 months; P=0.001) in angiosarcoma patients treated with gemcitabine.Conclusions:Our study suggests that higher hENT1 expression are associated to gemcitabine efficacy both in patients with advanced leiomyosarcoma and angiosarcoma.

Predictive value of hENT1 immunohistochemistry for gemcitabine-treated pancreatic cancer is antibody dependent.

e15746 Background: Pancreatic cancer (PC) continues to have poor outcomes, and biomarkers to guide individualized therapy are needed. Gemcitabine (GEM), a pyrimidine nucleoside, requires plasma membrane transporter proteins to enter cells, and deficiency of the human equilibrative nucleoside transporter 1 (hENT1) protein confers high-level GEM resistance in pre-clinical models. Furthermore, abundance of hENT in PC cells, as determined by immunohistochemistry (IHC), has been variably reported to be associated with improved survival in people with PC treated with GEM. The antibodies (Abs) used for IHC differ in targeted epitope, method of generation, and species of origin. The purpose of this review is to evaluate the contribution of anti-hENT1 Ab choice in finding a link between hENT1 expression and outcomes. Methods: We reviewed the published literature investigating relationships between hENT1 IHC tumor expression and survival in people with PC treated with GEM. Results: Eighteen studies were identified, using five distinct Abs (Table 1). We identified apparent differences in predictive value among Abs (Chi square test p = 0.065). All seven studies using the 10D7G2 Ab reported that high hENT1 expression is associated with significantly better outcomes in GEM-treated patients. Studies that used other anti-hENT1 Abs, in particular SP120 and 11337-1-AP, had lower rates of success. Pooled HRs and additional metrics will be presented. Conclusions: There is significant heterogeneity among studies evaluating the link between hENT1 expression and outcomes. hENT1 IHC with the 10D7G2 Ab is a strong and reproducible predictive marker for improved survival in people with PC treated with GEM. [Table: see text]

Predictive role of human equilibrative nucleoside transporter 1 in patients with pancreatic cancer treated by curative resection and gemcitabine-only adjuvant chemotherapy.

The predictive roles of human equilibrative nucleoside transporter 1 (hENT-1) in patients who undergo curative resection and adjuvant chemotherapy with gemcitabine alone have not been established. The present study retrospectively analyzed the clinical data from 101 consecutive patients who underwent curative resection and who received gemcitabine adjuvant chemotherapy for the treatment of pancreatic cancer at Kanagawa Cancer Center (Yokohama, Japan) between 2005 and 2014. The associations between the hENT-1 status and the survival and clinicopathological features of the patients were investigated. Of the 101 patients, 60 patients (59.4%) had high levels of hENT-1 expression. A significant association was observed between hENT-1 status and sex; however, for all the other clinicopathological parameters, including tumor and node stages, no differences were observed between the high and low hENT-1 expression groups. The median follow-up period of the present study was 67.3 months. Between the high and low hENT-1 expression groups, there was a statistically significant difference in the 5-year overall survival (OS) rates following surgery (20.6 and 8.9%, respectively; P=0.019). In addition, a significant difference was observed in the recurrence-free survival (RFS) rates at 5 years following surgery (P=0.049). hENT-1 status was one of the important predictive factors for OS and RFS in patients with pancreatic cancer who underwent curative resection followed by adjuvant chemotherapy with gemcitabine. Adjuvant chemotherapy with gemcitabine alone may be insufficient, particularly in patients with certain relevant risk factors.

АНАЛІЗ КОМБІНОВАНОГО ВПЛИВУ ІНТРАТУМОРАЛЬНОЇ ЕКСПРЕСІЇ ТИМІДИЛАТСИНТАЗИ ТА HENT1 НА ЕФЕКТИВНІСТЬ СХЕМИ ГЕМЦИТАБІН+ТЕГАФУР В АД’ЮВАНТОМУ ЛІКУВАННІ ХВОРИХ НА РАК ПІДШЛУНКОВОЇ ЗАЛОЗИ

Рак підшлункової залози (РПЗ) є одним із найагресивніших серед гастроінтестинальних раків. Шанс на довгострокову виживаність, що залишається неприйнятливо низькою, дає тільки радикальна резекція. Оптимізація ад’ювантного лікування хворих на РПЗ є актуальним питанням сучасної онкології. Метою роботи був аналіз комбінованого впливу інтратуморальної експресії hENT1 і тимідилатсинтази (TS) на ефективність схеми гемцитабін+тегафур ГемТег в ад'ювантному лікуванні хворих на РПЗ. До дослідження залучено 62 хворих на РПЗ з R0-cтатусом резекції на І та ІІ стадіях (UICC ver.7). Усі хворі отримували комбінацію ГемТег в якості ад’ювантної терапії. Діагноз у кожному випадку був веріфікований гістологічно, після чого імуногістохімічно визначали експресію hENT1 та TS. Статистичну обробку виконували за допомогою критерію Фішера, методів χі-квадрат, Каплана–Майєра та аналізу ризиків за Коксом. Медіана спостереження становила 34 місяці. За допомогою однофакторного аналізу оцінювали переваги у виживаності, порівняно з когортою хворих із співпаданням низької експресії hENT1 та TS. При цьому виживаність у когортах із високою експресією hENT1, з високою експресією TS та з високою експресією обох маркерів була значно вищою (р=0,053; р=0,03 та р&lt;0,001 відповідно). При багатофакторному аналізі ризиків за Коксом у стратифікованих когортах HR становив 4,65 (СI: 2,12–10,21), 4,82 (СI: 2,13–10,91) та 23,14 (СI: 2,73–195,88) відповідно. Висока інтратуморальна експресія hENT1 та TS незалежно одне від одного може покращувати загальну виживаність хворих на РПЗ при ад’ювантному лікуванні за схемою ГемТег. Хворі, в яких одночасно відзначалася висока експресія hENT1 та TS, отримували найбільшу перевагу в виживаності.

Differential expression of hENT1 and hENT2 in colon cancer cell lines.

Human equilibrative nucleoside transporters (hENT) 1 and 2, encoded by SLC29A1 and SLC29A2, permit the bidirectional passage of nucleoside analogues into cells and may correlate with clinical responses to chemotherapy in patients with colorectal cancer (CRC). The purpose of this study was to evaluate the expression profiles of SLC29A1 and SLC29A2 in human cancer cell lines. Using quantitative real-time polymerase chain reaction, we comprehensively profiled the transcription levels of SLC29A1 and SLC29A2 in 16 colon cancer cell lines. We validated the ubiquitous and heterogeneous distribution of SLC29A1 and SLC29A2 in human colon cancer cell lines and demonstrated that SLC29A1 was highly expressed in 25% of metastatic cell lines (Colo201 and Colo205) and 62.5% of primary cell lines (Caco2, Colo320, HCT116, RKO, and SW48). For the first time, we showed that both SLC29A1 and SLC29A2 were expressed at lower levels in colon cancer cell lines originating from metastatic sites than from primary sites. These findings indicate that most patients with metastatic CRC (mCRC) may have low hENT1 expression, and treatment with nucleoside analogues may be inefficient. However, some patients still show high hENT1 expression and have a high probability of benefiting from these drugs. Therefore, evaluating transporter expression profiles and different drug responses between primary and metastatic tumors in patients with mCRC is important. Further assessment of the association between hENTs and drug-based treatment of mCRC is required to elucidate the mechanisms of chemotherapy resistance.

P02 - Human equilibrative nucleoside transporter 1 as a predictor of efficacy to gemcitabine in advanced leiomyosarcoma and angiosarcoma

Background: The expression of human equilibrative nucleoside transporter 1 (hENT1), the known major gemcitabine transporter into cells, has been thoroughly investigated as a predictive marker of response to gemcitabine in pancreatic cancer and biliary tract cancers. Since gemcitabine is also widely used in the treatment of leiomyosarcoma and angiosarcoma, we decided to investigate the correlation between the expression of hENT1 and gemcitabine efficacy in these sarcoma subtypes. Material (patients) and methods: We retrospectively identified 71 patients affected by advanced angiosarcoma (26) or leiomyosarcoma (45) treated within five Italian referral centers for sarcoma, whose tumor tissue was available; among them, 49 patients (15 angiosarcoma, 34 leiomyosarcoma) were treated with gemcitabine as single agent in first line chemotherapy. All tumor samples were analyzed for hENT1 expression by Real Time PCR. Median 2-?Ct value was used as the cut off to dichotomize patients into “high” expression and “low” expression groups. Kaplan Meier analysis was performed to estimate Time to Progression (TTP) and Overall Survival (OS). The correlation between hENT1 expression and TTP/OS was ascertained using the log- rank test. Results: We found a significant association between high hENT1 expression levels and favorable outcome in terms of TTP and OS compared to cases with low hENT1 expression in leiomyosarcoma treated with gemcitabine (TTP: 6.8 vs 3.2 months, P= 0.004; OS: 14.9 vs 8.5 months, P= 0.007). Additionally, hENT1 overexpression correlated with a significant improvement in TTP (9.3 vs 4.5 months; P= 0.02) and OS (20.6 vs 10.8 months; P= 0.001) in angiosarcoma patients treated with gemcitabine. Conclusions: Our study suggests that hENT1 expression is a predictive marker of efficacy both in patients with advanced leiomyosarcoma and angiosarcoma who received gemcitabine monotherapy. A prospective clinical trial in a larger series of patients is warranted to confirm these results.

Abstract 449: Targeted proteomic analysis for personalized treatment of muscle invasive bladder cancer

Abstract Background: Standard treatment for muscle-invasive bladder cancer (MIBC) includes chemotherapy with either gemcitabine-cisplatin (GC) or methotrexate, vinblastine, adriamycin and cisplatin (MVAC). These regimens have similar clinical complete response rates of approximately 30%. While no targeted treatment has been approved for bladder cancer, clinical trials have identified biochemical markers that predict the chemoresponsiveness of MIBC tumors to specific chemotherapeutic agents. For example, patients with high hENT1 and low RRM1 expression responded better to GC-based chemotherapy than their counterparts, and HER2 overexpression predicted resistance to cisplatin-based therapy. To quantify targets that are known indicators of tumor behavior, we used targeted proteomic analysis to assess 30 different protein biomarkers in formalin-fixed paraffin-embedded (FFPE) MIBC tumor tissue. Methods: Twelve FFPE MIBC tissue blocks were obtained and a pathologist marked a minimum 8mm2 of tumor area from 1 or 2 slides. Following laser microdissection of the marked areas, proteins were extracted using the Liquid-Tissue® process and subjected to selected reaction monitoring mass spectrometry to quantify the amounts of 30 different targeted proteins in each patient sample. Results: Of the 12 patient samples, 7 (58%) expressed high levels of hENT1 and 11 (92%) expressed low levels of RRM1, indicating that gemcitabine-based therapy would be an appropriate choice. A single patient expressed high levels of RRM1, an indication for non-gemcitabine based therapy. All 12 patients expressed TUBB3, a marker of resistance to taxane (vinblastine) and 10 patients (83%) lacked expression of FR-alpha, a marker of sensitivity to methotrexate. The majority of patients expressed a marker of sensitivity to anthracycline (TOPO2A) and did not express a resistance biomarker for platinum therapy (ERCC1). Of 3 patients whose tumors expressed HER2, 2 had overexpression (&amp;gt;750 amol/ug) and would thus be eligible for HER2 basket trials. Further, multiplex-targeted proteomics discovered patients expressing FGFR1 (17%), cMET (33%), Axl (17%) and IDO1 (25%), which would make them eligible for clinical trials of targeted or immunotherapies. Conclusion: MIBC is heterogeneous and expresses a wide range of proteins. Yet, it continues to be treated with only 2 chemotherapeutic regimens. Multiplexed proteomics is currently being used in clinical practice to inform personalized patient care decisions with identification and the relative quantities of actionable proteins known to predict tumor behavior. Citation Format: Fabiola Cecchi, Sheeno Thyparambil, Adele Blackler, Todd Hembrough, Shahrooz Rabizadeh, Patrick Soon-Shiong, Henry Frierson, Daniel Theodorescu. Targeted proteomic analysis for personalized treatment of muscle invasive bladder cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 449.

High expression of the human equilibrative nucleoside transporter 1 gene predicts a good response to decitabine in patients with myelodysplastic syndrome.

BackgroundDespite the efficacy of decitabine treatment in myelodysplastic syndrome (MDS), no definite predictor of response is known. In this study, we investigated whether the expression levels of human equilibrative nucleoside transporter 1 (hENT1), hENT2, deoxycytidine kinase (DCK) and cytidine deaminase (CDA) genes could predict response to decitabine in MDS.MethodsWe performed quantitative real-time PCR in marrow mononuclear cells to examine the expression of hENT1, hENT2, DCK, and CDA prior to therapy in 98 MDS patients initially treated with decitabine. Response and overall survival of patients treated with decitabine were analyzed according to gene expression levels. HENT1 knockdown was performed by shRNA in the SKM-1 cell line, and the effect of this on the demethylation ability of decitabine on long interspersed nucleotide element 1 (LINE1) was investigated.ResultsPatients responding to decitabine presented with significantly higher hENT1 expression levels than non-responders (p = 0.004). Overall response, complete response, and cytogenetic complete response rate in patients with high hENT1 expression (79.4, 41.3, and 43.8 %) were significantly higher than those in patients with low hENT1 expression (48.6, 20.0, and 5.9 %, respectively) (p = 0.004, 0.033, and 0.006, respectively). In higher-risk MDS, patients with high hENT1 expression showed prolonged survival compared with those with low hENT1 expression (22.0 vs 14.0 months; p = 0.027). However, the expression levels of hENT2, DCK, and CDA did not affect response rate. Knockdown of hENT1 in SKM-1 cells weakened the demethylation effect on LINE1 induced by decitabine.ConclusionsHigh expression of hENT1 appears to predict a good response to decitabine and a prolonged survival in higher-risk MDS patients treated with decitabine. HENT1 expression knockdown weakens the demethylation effect of decitabine.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0817-9) contains supplementary material, which is available to authorized users.

Abstract 580: hENT1 testing in pancreatic ductal adenocarcinoma: Are we ready yet? A comparative analysis of the murine and the rabbit antibodies

Abstract Introduction Human equilibrative nucleoside transporter 1 (hENT1) level of expression in pancreatic adenocarcinoma (PAC) may predict survival in gemcitabine-treated patients after resection. These results have been obtained with a murine anti-hENT1 antibody (10D7G2), which is not commercially available. Another antibody, rabbit-derived (SP120), appears to have no predictive value in local, advanced or metastatic PAC. This raises the question of the equivalence of the antibodies. Patients and Methods Murine (10D7G2) and rabbit (SP120) monoclonal anti-hENT1 antibodies were applied on the same tissue microarrays of resected PAC. Two sets of patients from 2 separate institutions (n = 147 each) were used to compared hENT1 expression with both antibodies on two different immunochemistry automates. An additional amplification technique was performed on the second set. hENT1 expression was scored as previously reported. Results were correlated with overall survival (OS) following gemcitabine treatment. Results The rate of “hENT1 high” cases was lower with the SP120 clone (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, the concordance between the antibodies was found in only 50% of cases. High hENT1 expression was predictive of OS with the 10D7G2 antibody (HR = 0.49; 95% CI: 0.24-0.98; P = 0.045) but not with the SP120 (HR = 1.61; 95% CI: 0.80-3.21; P = 0.18). Conclusion Our data suggest that the two antibodies are not equivalent. Further studies are ongoing and will be presented at the meeting: 1/ Comparative analysis of the rabbit antibody on a larger series (n = 471) with staining performed by Ventana® (Roche®), 2/ Comparative analysis of 3 additional anti-hENT1 antibody together with mRNA level correlation, 3/ Comparative expression of hENT1 in a series of matched primary tumors-metastases. Citation Format: Jerome Cros, Raphael Marechal, Jean Baptiste Bachet, Pierre Laurent-Puig, Jean Francois Flejou, Pieter Demetter, Magali Svrcek. hENT1 testing in pancreatic ductal adenocarcinoma: Are we ready yet? A comparative analysis of the murine and the rabbit antibodies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 580. doi:10.1158/1538-7445.AM2015-580

The association of epithelial expression of hENT1 and survival from adjuvant gemcitabine in pancreatic ductal adenocarcinoma (PDAC).

296 Background: Human equilibrative nucleoside transporter 1 (hENT1) mediates the cellular uptake of gemcitabine. Low tumour hENT1 expression has been associated with reduced survival from gemcitabine in some studies. We analyzed the prognostic effect of hENT1 expression in a cohort of PDAC patients (pts) who underwent surgical resection. Methods: A tissue microarray (TMA) composed of duplicate 0.6 mm cores from 261 resected PDACs was constructed from all available cases derived from the archives of Vancouver General Hospital between 1988 - 2013. TMA sections (4 µm) were stained with an antibody for hENT1 (SP120- Spring Biosciences) and scored using a four-tiered scoring system (negative, weak, moderate and strong staining). The expression of hENT1 in the epithelial component of PDAC was quantified and patient samples were divided into low (negative and weak staining) and high (moderate and strong staining) hENT1-expressing groups. Treatment and survival data were collected. Survival differences were quantified with Kaplan-Meier method and log-rank statistic. Results: Our cohort included pts who received no adjuvant chemotherapy (CT) (n=182) or gemcitabine (n=59). Cases treated with other regimens (n=20) were excluded. There was no significant difference in the median overall survival (OS) between pts with low (14.0 months, n=167) and high hENT1 expression (15.2 months, n=15) in the absence of adjuvant CT. Gemcitabine treated patients with low hENT1 expression (n=51) had a median OS of 20.9 months. Median OS was not reached in the group of pts with high hENT1 expression (n=8) who received gemcitabine, with only 2 deaths at the 24 month time point and 6 censorings (p = 0.045). Pts who received gemcitabine had an increased median OS (24.8 months) compared to pts who did not receive adjuvant CT (14.0 months) regardless of hENT1 expression (p&lt;0.0001). Conclusions: Consistent with the ESPAC-3 data, in our analysis adjuvant gemcitabine was associated with increased OS in PDAC pts with high epithelial hENT1 expression (determined using the SP120 antibody) compared to pts with low hENT1 expression. Prospective evaluation of the predictive value of hENT1 expression in this setting is needed.

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

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. 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). The two antibodies are not equivalent. Further prospective studies seem to be warranted before hENT1 testing for PDAC is used in daily practise.