Background
Biliary tract cancer (BTC), arised from the epithelial cells of the biliary tract, encompassing malignant neoplasms of gallbladder and bile duct [
1]. BTC comprises about 3% of gastrointestinal tumors and the incidence of it seems to be increasing [
2]. Although a number of risk factors and molecular mechanism for BTC have been found, the 5-year survival is still as low as 10% [
2]. Gallbladder cancer (GBC) is the most common biliary tract malignancy and the seventh most common gastrointestinal cancer, with high mortality rate owing to the early infiltration of the tumor cells into the liver by lymphatic, perineural and hematogenous routes [
3]. Extrahepatic bile duct cancer (EBDC) consists of hilar and distal bile duct carcinoma, with a poor 5-year survival rate ranging from 20%–45% [
4]. In spite of GBC and EBDC originate in different anatomic biliary tree, there are still overlapping etiologic risk factors, patients characteristics, histopathology and molecular biology. Complete resection is the only potentially curative therapy for patients with biliary tract cancer; however, biliary tract tumors are usually asymptomatic at early stage and the majority of patients present with advanced, unresectable tumors [
5]. Therefore, surgery alone is not sufficient treatment for biliary carcinoma, especially in advanced stages. And the adjuvant therapeutic modalities, such as chemotherapy or radiotherapy, are needed to improve long-term survival [
6].
Although recent BILCAP study have reported capecitabine could extend survival in UK patient, there is still no established adjuvant chemotherapy other than several attempts to identify effective agents for patients with biliary cancer in Asia [
6]. According to NCCN clinical practice guideline of hepatobiliary cancers, gemcitabine-based adjuvant chemotherapy is recommended for biliary tract cancer patients. However, no prospective studies have demonstrated the efficacy of adjuvant gemcitabine for patients with biliary carcinoma [
7]. Therefore, there is an urgent need for identifying a precise biomarker to better predict patient benefitting from adjuvant chemotherapy.
Tumors contain cancer cells and recruit normal cells, creating the tumor microenvironment, including innate immune cells (neutrophils, macrophages, dendritic cells, natural killer lymphocytes, mast cells) and adaptive immune cells (T and B lymphocytes) [
8]. Both innate and adaptive immune systems have been implicated in promoting and preventing tumor growth. TIMs are attracted by tumor microenvironment by stem cell factor (SCF) secreted by tumor cells and secrete several angiogenic factor as well as matrix metalloproteinases (MMPs), which promote tumor vascularization and invasiveness, respectively [
9,
10]. On the contrary, TIMs exert inhibition of tumor cell growth through releasing IL-1, IL-4, IL-6, IL-8, monocyte chemotactic protein-3 and -4 (MCP-3 and MCP-4), transforming growth factor beta (TGF-β), and chymase [
10]. TIMs in prostate cancer, [
11] metastatic bladder cancer, [
12] Hodgkin’s lymphoma, [
13] thyroid cancer, [
14] pancreatic cancer, [
15‐
17] metastatic colorectal cancer, [
18] and non-small cell lung cancer [
19] were associated with poor prognostic outcome,whereas TIMs in local colon cancer, nonmetastatic and invasive breast cancer confer a favorable prognosis [
20,
21]. A recent research reported TIMs participate in the progression and metastatic potential of cholangiocarcinoma [
22]. However, the effect of peritumoral TIMs for biliary tract cancer and the precise function mechanisms in tumor progression still remain obscure. Furthermore, few studies have demonstrated a relationship between TIMs and clinical outcomes of patients with biliary tract cancer.
In this study, we explored whether TIMs could predict the survival of GBC and EBDC patients who received gemcitabine-based chemotherapy after operation by immunohistochemical staining and evaluated its correlation with clinicopathological characteristics.
Methods
Patients selection
A total of 250 consecutive patients with GBC or EBDC, including 164 with GBC, 64 with perihilar cholangiocarcinoma (PHC) and 22 with distal cholangiocarcinoma (DC) underwent surgical resection between May 2004 and April 2012 at Zhongshan Hospital, Fudan University (Shanghai, China) were recruited in present study. Ampullary tumors were excluded from the EBDC due to potential different biology. The total 250 patients were assigned into two independent patient cohorts: discovery set (n = 115) and validation set (n = 135) according to different follow-up time. Of these patients, 219 received radical resection (R0), 100 received gemcitabine-based chemotherapy after surgical resection (at least one cycle). This study was approved by the Ethics Committee of Zhongshan Hospital, and written informed consent was obtained from all patients. The clinicopathological and baseline demographic characteristics of the patients including age, gender, tumor location (PHC, DC or GBC), tumor differentiation, vascular invasion and TNM stage were collected retrospectively. OS was calculated from the date of surgery to the date of death or censored at the date of last visit. The median follow-up time of the patients after operation was 26.7 months, 11.7 months for discovery set and validation set, respectively.
Tissue microarray and immunohistochemistry
Tissue microarray (TMA) was established with formalin-fixed paraffin-embedded surgical specimens, and immunohistochemical staining was performed on TMA according to the protocols previously described [
23] with appropriate antibodies after control staining (Mouse anti-tryptase monoclonal antibody, diluted 1:1000; Abcam, Cambridge, UK; anti-CD8, IR623, DAKO, ready-to-use). The negative control sections were treated equally with primary antibody omitted. The number of MCs and CD8 + T cells per field was evaluated with Image pro plus 6.0 (Media Cybernetics Inc., Bethesda, MD). Identical settings were used for each photograph. Positive staining were calculated under high magnification field (HPF, 400×). The intensity of MCs or CD8 + T cells was scored as the mean number of MC or CD8 + T cell positive/HPF from entire filed independently by two pathologists. The cut-off point for the high/low MCs or CD8 + T cells infiltration was determined with the X-tile software. Overall survival curves were plotted by the Kaplan-Meier method, and log-rank test was used to analyze the difference between subgroups.
Statistical analyses
Statistical analysis was performed with SPSS 22.0 (IBM Corporation, Armonk, NY, USA), Medcalc Software (version 15.2.2; Medcalc, Mariakerke, Belgium) and Stata SE, version 13.0 (Stata, College Station, TX). Chi-square test or Fisher exact test was used to evaluate the correlation between clinicopathological features and immunohistochemical variables. Continuous variables were analyzed by means of t test. Univariate and multivariate regression analysis was performed with Cox proportional hazards regression model. To further explore the prognostic value between TIMs density and clinical outcomes, we applied Kaplan-Meier analysis to compare overall survival (OS) between subgroups. The log-rank test was used to compare survival rates. All tests were two-sided, and P < 0.05 was regarded as statistically significant.
Discussion
A large number of studies have tried to identify the contributory functions of tumor associated mast cells in tumor growth [
16]. In the studies about colon, [
27] gastric, [
28] pancreas, [
29] bladder cancers, [
12] mast cells appear to be pro-tumorigenic and are associated with poor prognosis. However, Welsh et al. demonstrated that in non-small-cell lung cancer, tumor cell islet mast cells had a strong independent association with an improved prognosis and the stromal mast cells number was not significantly associated with survival [
23]. In contrast to above studies, we observed that TIMs was mainly located in tumor stroma tissues and was positively correlated with prolonged overall survival. These results indicated that TIMs, which is modified by their environment, could display different phenotype and thus exert beneficial or detrimental effects on tumor progression. Previous study about hepatocellular carcinoma reported that peritumoral mast cells play a critical role in the suppression of immune reactions of tumors and cooperate with Tregs to sustain allograft tolerance and exacerbate tumor immunosuppression [
30]. Nakae et al. demonstrated that mast cells activate T cells via TNFα release and through cell-cell interactions via OX40L [
31]. Considering the role of mast cells plays in the developing tumor, we proposed that mast cells might present an anti-tumor effect through activating cytotoxic T cells and inducing an immune-stimulating environment in this study. Spearman’s correlation analysis indicated that TIMs stromal infiltration was positively correlated with anti-tumor CD8+ T cells. These findings confirmed the hypothesis that TIMs may play a critical role in activating and promoting CD8+ T cells to reject tumors. Several previous studies of inflammation suggested that activated mast cells facilitate anti-infection by enhance recruitment of NK and CD8+ T cells [
31,
32]. Sharon A. Oldford et al demonstrated that mast cells could display antitumor activity by recruiting CD8+ T cells via secreting CCL3 in the tumor context [
33]. The stromal infiltration of CD8+ T cells in tumor context is the basis for patients who receive immunotherapy. Accordingly, TIMs might serve as an important prognostic factor in identifying biliary tract cancer patients for the combination of chemotherapy and immunotherapy.
Gemcitabine-based ACT is an important modality for patients with advanced biliary tract cancer after surgery. More and more recent studies demonstrated that administration of gemcitabine or combined with platinum could significantly prolong survival of patients with advanced biliary tract cancer [
34‐
36]. However, few researches focused on identifying patients whose tumor will be sensitive to ACT [
7]. Therefore, we compared the OS of patients who did or not receive gemcitabine-based ACT and found that those who suffered from high TIMs stromal infiltration could significantly benefit from gemcitabine-based ACT. Considering TIMs in biliary tract cancer were positively associated with CD8+ T cells, it suggested that high TIMs infiltration correlated with an immune-stimulated microenvironment and inhibited tumor progression via recruitment of antitumor immune cells. These findings indicate that TIMs could be a vital factor for predicting chemotherapeutic response, which could be valuable for selection and management of patients who receive ACT. Therefore, it is essential to stratify that if patients could benefit from ACT and avoid the excessive toxicities of ACT.
The study has limitations that it is a retrospective single institution research and sample size is relatively small. Moreover, the results may not be generalizable to other populations and to patients treated with adjuvant capecitabine. In addition, the results were based on immunohistochemistry of tissue microarrays, which is a semiquantitative method and may not be typical. A prospective, larger, multi-centered randomized trial is required to validate these findings in future.
Acknowledgements
The authors would like to thank Dr. Rongkui Luo, Dr. Lingli Chen (Department of pathology, Zhongshan hospital of Fudan University) for diagnosis confirmation and technical assistance, respectively.