Annals of Oncology Advance Access originally published online on January 10, 2008
Annals of Oncology 2008 19(4):724-728; doi:10.1093/annonc/mdm576
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gastrointestinal tumors |
Human equilibrative nucleoside transporter 1 (hENT1) protein is associated with short survival in resected ampullary cancer
1 Department of Medical Oncology
2 Department of Surgical Pathology, University Campus Bio-Medico, Rome, Italy
3 Department of Oncology, Cross Cancer Institute, University of Alberta, Edmonton, Alberta, Canada
4 Department of Surgery, University Campus Bio-Medico, Rome
5 Department of Surgery
6 Department of Surgical Pathology, Catholic University of the Sacred Heart, Rome
7 Department of Biomedical Researches, Campus Bio-Medico University, Rome, Italy
* Correspondence to: Dr D. Santini, Department of Medical Oncology, University Campus Bio-Medico, via Emilio Longoni 81, 00155 Rome, Italy. Tel: +39-06-22541739; Fax: +39-06-22541520; E-mail: d.santini{at}unicampus.it
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Abstract |
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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.
Key words: ampullary cancer, hENTI, survival
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introduction |
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Although carcinoma of the ampulla of Vater is an uncommon entity, it accounts for 20%–40% of the resected cases of all periampullary neoplasms [1–3]. Many studies have examined the outcomes of radically resected ampullary cancer, but most studies had insufficiently large sample sizes to assess prognostic factors, and in larger series there remains substantial differences in the main independent prognostic variables. At present, the most reliable prognostic variable in resected ampullary cancer is the presence and number of lymph nodes with metastatic deposits, and no molecular prognostic factors have been validated in this disease [4, 5].
Similarly, the adjuvant therapy for resected ampullary carcinoma is poorly studied due, in part, to the rarity of the cancer. Case series of adjuvant chemoradiotherapy, generally infusional fluorouracil with concurrent radiotherapy [6, 7], achieve median survivals on the order of 3 years. Furthermore, there is no generally accepted chemotherapy for those with recurrent or distant metastatic disease.
Gemcitabine (Gemzar, Eli Lilly, Indianapolis, In) is a pyrimidine nucleoside analogue that is the most single effective agent in the chemotherapy of pancreatic adenocarcinoma [8]. Moreover, gemcitabine appears to be a reasonable alternative to best supportive care in the treatment of advanced biliary tract cancers, on the basis of encouraging results in nonrandomized clinical studies [9, 10]. Because the biochemical targets of gemcitabine are intracellular, the mandatory first step in the production of cytotoxicity is permeation across the plasma membrane. Gemcitabine is a hydrophilic molecule and does not cross the plasma membrane by diffusion; efficient cellular uptake requires the presence of specialized plasma membrane nucleoside transporter (NT) proteins [11]. The human equilibrative nucleoside transporter 1 (hENT1) is a ubiquitous protein and is the major means by which gemcitabine enters cultured human cells and hematopoietic progenitor cells [12]. The abundance and distribution of the hENT1 protein can be evaluated using immunohistochemistry and has been assessed in a number of malignant and benign tissues [13–16]. Of potential relevance, immunohistochemical variations of hENT1 in pancreatic adenocarcinoma samples significantly correlate with median survivals after gemcitabine therapy in patients with advanced disease, and has recently been shown to predict benefit from gemcitabine chemotherapy in the setting of resected pancreatic cancer [17, 18].
On the basis of these findings, we studied the abundance and distribution of hENT1 in tumor samples from 41 patients with radically resected cancer of the ampulla and correlated these biological results with clinical parameters and disease outcomes.
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materials and methods |
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clinical data and tumor specimen acquisition
This retrospective study was restricted to patients with ampullary carcinoma and was consecutively treated at the Catholic University School of Medicine of Rome and at the University Campus Bio-Medico of Rome from 1986 to 2005. To be eligible for this analysis, each subject underwent surgical resection for tumors of ampullary origin with curative intent, and only patients without known residual disease were analyzed. All patients were staged before surgery by clinical examination, computed tomography of the thorax, abdomen, and pelvis, and, when indicated, intraoperative ultrasound of the liver, excluding the presence of overt distant metastases. Data on clinical variables, including sex, age, preoperative assessment of disease state, and type of operative procedure, were gathered retrospectively from patient records. All specimens underwent gross anatomical examination according to the procedure described by Rosai [19] including evaluation of all anatomic structures (pancreatic duct, ampulla of Vater, common bile duct, and pancreatic head). All tumors included in the study were limited to the ampulla or were primarily located in the ampulla, and secondarily spreading into the neighboring structures. Pathological findings (tumor size and spread, and lymph node status) were obtained from the pathologists’ original reports. In addition to the original pathological reports, TNM (tumor–node–metastasis status) classification was reassessed as International Union Against Cancer [20]. Survival was determined from the date of initial surgery to the date of death or the last contact. Follow-up data were available for all included patients. The study was carried out with approval of the relevant local institutional research boards.
tissue preparation and immunostaining
Representative tumor blocks were sectioned at 3-µm thickness for immunohistochemical studies. The sections were deparaffinized with three immersions in xylene baths (10 min each) followed by serial washes in graded alcohol from 100% to 50%. After rinsing in water, slides were placed in 250 ml of high pH 1x DAKO target antigen retrieval solution and microwaved in TT-mega Milestone (ESBE Scientific, Markham, Ontario, Canada) under controlled temperature and high pressure for 10 min at 100°C. After cooling in water for 6 min, slides were rinsed with water and peroxidase blocked in 3% hydrogen peroxide solution with methanol for 10 min then washed in running water for 10 min. Phosphate-buffered saline (PBS) (pH 7.2) was used for rinsing before incubation in a humidified chamber overnight at 4°C with appropriate dilutions of anti-hENT1 mouse mAb developed and characterized as described previously [13, 21] and anti-Ki-67 mouse mAb (clone MIB-1, DAKO, Cytomation Inc., Carpinteria, CA). The sections were then rinsed with PBS, immersed in buffer for 5 min, and incubated with goat anti-mouse dextran conjugate (DAKO Envision) for 30 min followed by soaking in PBS. DAKO diaminobenzidine liquid chromagen was placed on the samples for 5 min and rinsed, after which, the slides were soaked in 1% CuSO4 for another 5 min. Subsequently, the sections were rinsed, counterstained with hematoxylin, dehydrated through graded alcohol and xylene, and finally coverslipped. Negative controls were provided by omitting the primary antibodies. Immunostaining was assessed by two independent pathologists blinded to clinical characteristics and outcomes.
Scoring for hENT1 was on the basis of relative intensities of staining of the ampullary tumor with reference to the normally present hENT1 staining of cell membranes within the islets of Langerhans’ cells and lymphocytes. These internal references were then used as internal positive controls between slides and samples as well as for the staining procedure. Ampullary tumor tissue was then evaluated by comparison with the internal controls. Staining intensity was graded as absent (0), positive but less intense than internal control tissue (1+), positive-like internal control tissue (2+), and positive, more intense than internal control tissue (3+). Samples with regions of varying staining intensities of hENT1 were scored and the percentages of each staining intensity were recorded. Finally, tumors with an intensity staining of 2+ and 3+ in 
90% of the tumor cells were considered as high expression of hENT1 [17].
Ki-67 staining was carried out as previously reported [22] and evaluated by counting the number of positively stained nuclei in a total of at least 1000 tumor cells. Immunoreactivity for Ki-67 was categorized by the percentage of nuclear staining.
statistical analysis
A univariate survival analysis for each prognostic variable on overall survival (OS) was estimated as by the Kaplan–Meier method [23]. The terminal event was death, attributable to cancer or noncancer causes. The statistical significance of the differences in survival distribution among the prognostic groups was evaluated by the log-rank test [24]. The Cox proportional hazards model was applied to the multivariate survival analysis [25]. The prognostic variables for OS included age, gender, T and N factors, hENT1 expression, and Ki-67 staining. Spearman’s rank test was applied to establish a correlation between hENT1 expression and tumor local extension (T factor), nodal status (N factors). Pearson correlation test was used to establish the difference between hENT1 expression and Ki-67. P values <0.05 were regarded as statistically significant in two-tailed tests. SPSS software (version 13.00, SPSS, Chicago, IL) was used for statistical analysis.
patient characteristics
The main clinicopathological features are summarized in Table 1. The cohort consisted of 41 patients with pathological diagnosis of radically resected cancer of the ampulla (23 men and 18 women). The median age at diagnosis was 63 years (range, 38–78). Seven patients (17.1%) were classified as T1, 17 (41.5%) as T2, 15 (36.6%) as T3, and only 2 (4.9%) as T4. Eighteen (43.9%) patients had locoregional lymph node metastasis. Adjuvant radiotherapy and/or chemotherapy for ampullary cancer was not routinely offered in the hospitals involved in the study. The median duration of follow-up after surgery was 55 months (range, 6–110). The median OS was 45 months (range, 6–100). The 1-, 3-, and 5-year OSs were 87.8%, 29.2%, and 26.8%, respectively. After a median follow-up of 55 months, 24 patients (58.5%) are still alive without evidence of disease, 3 patients (7.3%) are alive with recurrence of disease, and 14 patients (34.1%) are dead (10 patients died of ampullary carcinoma and 4 of noncancer-related causes).
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clinicopathological correlation
A positive statistical correlation was found between T and N factor (P = 0.001).
hENT1 and Ki-67 staining
The immunohistochemical hENT1 staining was present within the islets of Langerhans' cells and lymphocytes, as previously reported, and were used as internal positive controls. Within carcinoma cells, hENT1 protein staining was localized to the cytoplasm. The peritumoral stroma cells were otherwise negative. Of the 41 tissue samples, 12 (29%) samples had uniformly high hENT1 immunostaining (intensity scores of 2+ and/or 3+) with no heterogeneous regions with low or absent hENT1 expression. Twenty-nine (71%) samples possessed a proportion (range, 11%–100%) of adenocarcinoma cells without or with a low detectable hENT1 (intensity scores of 0 and/or 1+) (Figure 1).
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Immunohistochemical staining of tumors with anti-Ki-67 antibodies showed a primarily nuclear localization. The median value of Ki-67 percentage in all studied tumors was 40% [interquartile range (IQR) 20%–50%]. Furthermore, median value of Ki-67 in low and high hENT1 expression group was, respectively, 30% (IQR 5%–40%) and 50% (IQR 47.5%–52.5%). Pearson correlation test showed a statistically 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 TNM).
immunohistochemistry, clinicopathological variables, and patient survival
To determine the prognostic role of hENT1 and Ki-67 protein expression in a univariate survival analysis, patients were stratified according to the dichotomized variables (criteria as stated above) in positive (hENThigh expression and Ki-67>20%) versus negative (hENTlow expression and Ki-67<20%). The main clinicopathological patient features (gender, age, and T and N factors) were included in the univariate analysis. On univariate analysis, OS was not associated with traditional clinicopathological features (Table 2).
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Both the molecular factors analyzed, hENT1 and Ki-67 expression, however, were associated with 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; Table 2).
The median survival in patients with high hENT1 expression was only 41 months, as compared with 77 months in those with low hENT1 expression (P = 0.022). The median survival time in patients with high Ki-67 staining was 45 versus 80 months in those with low Ki-67 staining (P = 0.05) (Table 2). All variables that reached significance in the univariate survival analysis were incorporated in the multivariate analysis, with disease-related survival used as primary end point. In this model, neither hENT1 expression nor Ki-67 staining was defined as an independent prognostic factor (data not shown).
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discussion |
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There are few substantial data reporting significant prognostic markers for ampullary cancer patients. The influence of pathological (tumor size, lymph node involvement, status of resection margins, and perineural invasion), surgical (aggressive surgical approach versus limited resection), and biological (p53, c-erbB2, bax-2, mapsin, and apoptotic index) factors is not clear [26–29].
There is substantial interest in identifying and validating molecular markers to select patients with a high likelihood of benefiting from specific chemotherapy regimens. Gemcitabine, a nucleoside chemotherapy drug used in many solid tumors, requires the NT protein hENT1 to efficiently enter cells [30], and hENT1 deficiency confers resistance to gemcitabine in vitro [12]. Recently, the immunohistochemical assessment of the NT protein hENT1 has shown both prognostic and predictive value in the setting of adenocarcinoma of the pancreas treated with the nucleoside chemotherapy drug, gemcitabine [17, 18]. For these reasons, we characterized the pattern of expression and to explore the possible prognostic significance of hENT1 expression in a homogeneous cohort of patients with radically resected ampullary cancer.
Similar to the previous description of hENT1 in pancreatic carcinoma [17], immunohistochemical identification of the hENT1 protein in ampullary cancer tissue revealed two patterns of staining, either (i) uniformly positive staining of adenocarcinoma cells or (ii) heterogeneous regions lacking hENT1 in a proportion of adenocarcinoma cells (range, 10%–100%). Internal controls for this assessment were provided by positive staining (islet of Langerhans’ cells and lymphocytes).
In this cohort of patients, we demonstrated that about one-third of the samples had uniformly high hENT1 immunostaining (intensity scores of 2+ and/or 3+). This finding indicates that one-third of patients may have a higher probability to benefit from gemcitabine-based chemotherapy. Further studies with gemcitabine-treated ampullary carcinoma assessed for hENT1 immunostaining will, however, be required to evaluate this hypothesis.
Interestingly, we demonstrated a strong positive correlation between hENT expression and Ki-67 score. This positive correlation indicates that hENT1 protein levels may be correlated with the proliferation rate of malignant cells. This finding is consistent with previous reports indicating an association between hENT1 and proliferative rate of lymphoproliferative malignancies [31]. The regulation of hENT1 RNA expression and protein levels is poorly understood. hENT1 is variably expressed within normal tissues and malignant cells. Various factors that influence hENT1 expression have been identified and include hypoxia [32, 33] and differentiation status [34].
This study clearly showed that high immunohistochemical staining for the hENT1 protein is significantly correlated with poor prognosis in patients with resected cancer of the ampulla of Vater. In the multivariate analysis, neither hENT1 expression nor Ki-67 staining have showed to reach the statistically significance as an independent prognostic factor, probably due to the small sample size of patients with this relatively rare tumor type included in the study. Given the positive association between hENT1 protein and benefit from gemcitabine in pancreatic cancer, this population might particularly derive benefit from gemcitabine-based therapy. These findings warrant the consideration of prospective trials of gemcitabine in resected ampullary carcinoma, with tissue assessment to define the relationship between hENT1 immunohistochemical staining and clinical outcomes. Furthermore, gemcitabine may be of particular benefit in those patients with metastatic or recurrent disease expressing high levels of hENT1 in tumor samples.
In conclusion, immunohistochemistry for the hENT1 protein carries prognostic information in patients with resected ampullary cancer and holds promise as a predictive factor to assist in chemotherapy decisions.
Received for publication October 22, 2007. Revision received November 15, 2007. Accepted for publication November 20, 2007.
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