hTERT Represents an Innovative Bio-marker in Cholangiocarcinoma Detection

Background: The study was aimed to examine the diagnostic value of serum human telomerase reverse transcriptiptase (hTERT) in patients with cholangiocarcinoma (CCA). Methods: Serum hTERT in CCA patients and healthy controls was detected by quantitative real-time PCR (qRT-PCR) and compared by student’s t-test. Relationships of serum hTERT and clinical parameters were explained by Chi-square test. The receiver operating characteristics (ROC) analysis was conducted to identify the diagnostic value of serum hTERT in CCA. Results: Serum hTERT was up-regulated in CCA patients compared with healthy controls (P<0.05). Furthermore, serum hTERT was closely related with differentiation (P=0.005), distant metastasis (P=0.015) and TNM stage (P=0.009). The AUC value of ROC curve was 0.901, demonstrating that serum hTERT could discriminate between CCA and healthy individuals, with the cutoff point of 1.88. Besides, the diagnostic specicity and sensitivity of serum hTERT in CCA were 89.7% and 76.0%, respectively. Conclusions: hTERT may be a promising biomarker for diagnosis of CCA.


Background
Cholangiocarcinoma (CCA) is a rare malignancy in the intrahepatic or extrahepatic biliary tree, originating from the epithelial cells of bile ducts [1,2]. According to the anatomical and clinical criteria, CCA is classi ed into three subtypes: intrahepatic CCA, extrahepatic CCA and hilar CCA [3,4]. Surgical resection strategy remains the only potential curative treatment for CCA patients [5,6]. However, the major CCA cases are usually diagnosed with disseminated or advanced stage that miss the optimal time for operation, leading to poor outcomes [7]. Although the symptoms of advanced CCA include biliary strictures, abdominal pain, jaundice and fever, even anorexia, vomiting and nausea, the cancer is asymptomatic at early stage, increasing the di culty for early diagnosis [8,9]. So far, several biomarkers are identi ed for CCA screening, such as CEA, CA19-9, ALP, MUC5AC, and CA-S121. However, their clinical value is limited by low diagnosis accuracy [10]. Therefore, it is urgently needed to explore novel biomarker for CCA diagnosis.
Telomerase, a type of ribonucleoprotein, locates at the ends to the chromosomes and consists of two subunits: telomerase RNA template (hTR) and human telomerase reverse transcriptiptase (hTERT) [11].
Telomerase plays a pivotal role in cell division via catalyzing the synthesis of telomeric repeat DNA [12,13]. hTERT, a catalytic subunit of telomerase, can regulate the activity of telomerase [14,15]. hTERT is undetectable in normal physiological conditions, however, its over-expression has been observed in various human cancers, including gastric cancer [12], hepatocellular carcinoma [15], and colorectal cancer [16]. Furthermore, its up-regulation shows signi cant association with aggressive potential of cancer cells, suggesting is promoting function in tumor development and progression [17]. For example, Liang et al. reported that patients with endometrial cancer showed signi cantly higher blood hTERT level than the healthy individuals, moreover, its expression pattern was positively associated with tumor stage, revealing its predictive function for tumor initiation [18]. The elevated expression of hTERT was also reported in CCA, however, its diagnostic value for the disease remained unidenti ed [19].
In the present study, we attempted to detect the serum level of hTERT in patients with CCA and estimate its diagnostic value for this disease.

Methods
Patients and specimens A total of 107 CCA patients at the PLA Rocket Force Characteristic Medical Center were enrolled in this study. All the recruited patients were pathologically diagnosed by two physicians. None of them had received chemo-or radio-therapy 3 weeks before investigation. The clinical information of patients, such as age, gender, differentiation and TNM stage, was listed in Table 1. In addition, 75 healthy donors in the same hospital were recruited as healthy controls. Serum samples were isolated from peripheral blood of both CCA patients and healthy controls via centrifugation. This study was approved by the Ethics Committee of the PLA Rocket Force Characteristic Medical Center, and all patients had provided the informed contents in advance.
Quantitative real-time polymerase chain reaction PCR (qRT-PCR) Total RNA was isolated from serum samples using Trizol RNA extraction kit (Invitrogen) according to the manufacture's instructions. Then total RNA was used to synthesize the rst strand of cDNA using the iScript cDNA synthesis Kit (Bio-Rad, USA). The relative mRNA level of hTERT was evaluated by qRT-PCR.

Statistical analysis
All data were analyzed in SPSS18.0 software (SPSS, Inc., Chicago, IL, USA). Student's t-test was used to compare the expression difference of serum hTERT between groups. The association of hTERT expression and clinical parameters was validated by χ 2 test. Receiver operating characteristics (ROC) curve was plotted to assess the diagnostic value of serum hTERT in CCA. The results were considered to be signi cant when P value was less than 0.05.

Results
Up-regulation of serum hTERT mRNA in CCA patients Serum hTERT mRNA levels were determined in 107 CCA patients and 75 healthy controls using qRT-PCR. The expression of serum hTERT mRNA in CCA patients was signi cantly higher than that in healthy controls was (P<0.001) (Figure 1).

Association between hTERT expression and clinical parameters
The patients were divided into two groups according to the median level of serum hTERT mRNA: high expression group and low expression group. Chi-square test demonstrated that the expression pro le of hTERT showed close link with differentiation (P=0.005), distant metastasis (P=0.015) and TNM stage (P=0.009) ( Table 1). However, no obvious correlation was detected between serum hTERT expression and age, CA19-9 level, or gender (P>0.05 for all).
Diagnostic performance of serum hTERT in CCA The diagnostic accuracy of serum hTERT in CCA was determined by ROC analysis. As shown in Figure 2, the AUC value of the curve was 0.901, suggesting that serum hTERT could discriminate between CCA patients and healthy individuals. The diagnostic sensitivity and speci city of serum hTERT in CCA were 89.7% and 76.0%, respectively, with the optimal cutoff value of 1.88. Discussion CCA is a fatal disease of bile duct epithelial cells, with dismal prognosis [20]. Due to the delay in early diagnosis, the major of CCA patients are diagnosed with metastatic disease, leading to limited therapeutic effects and poor outcomes [21]. Thus, identi cation of novel biomarkers for early diagnosis of CCA may be a promising way to improve the prognosis of CCA patients. Like other kinds of tumors, the pathogenesis of CCA is a complex process, with the involvement of various molecules. In the previous studies, number of molecular biomarkers were identi ed for CCA. In the study of Khoontawad et al., EXT1 was proved to be overexpressed in CCA and showed close correlation with CCA genesis, suggesting its potential as a biomarker for the disease [22]. Tang  positively correlated with aggressive progression of CCA that might be a candidate biomarker for diagnosis and treatment strategies of the disease [24]. To explore the molecular biomarkers for CCA early detection and prognosis evaluation may be an effective approach to improve the outcomes of the patients. In the current study, we investigated the prognostic signi cance of serum hTERT for CCA.
The molecular abnormalities in tumorigenesis are mainly involved in independent growth, unlimited replication, aging avoidance, block of growth inhibitory signal and escape of apoptosis. Telomeres play an important role in maintaining chromosome stability and cell activity in different animal cells. In normal cells, telomeres are usually presented with procedural shortening, resulting in the apoptosis and senescence of cells. However, in the immortalized and tumor cells, the telomere lengths are maintained by the activated telomerase, resulting the in nite proliferation and canceration [25]. hTERT, a subunit of telomerase, plays a crucial role in telomerase activity mainly [26,27]. Overexpression of hTERT has been found in a large number of cancers, as well as cholangiocarcinoma [19,28]. Furthermore, its expression patterns showed close association with development and progression of cancer. Based on this, we deduced that hTERT might hold the potential to serve as a biomarker for CCA.
In the present study, we investigated the expression of serum hTERT mRNA in CCA patients and healthy controls using qRT-PCR. The results showed that serum hTERT was increased in CCA patients compared with healthy controls. Furthermore, the elevated expression of hTERT was positively associated with poor tumor differentiation, positive distant metastasis and advanced TNM stages. All the data revealed that hTERT played a carcinogenic role in development and progression of CCA. The conclusion was consistent with the previous study [19]. It was reported that hTERT might regulate tumor metastasis via miR-29a-ITGB1 pathway [29]. A related research also reported that hTERT promoted cancer invasion and metastasis through coorperating with c-Myc to upregulate the expression of heparanase [12]. However, the speci c oncogenic mechanisms for hTERT in CCA remained unidenti ed. Further researches were still needed.
In addition, ROC analysis was established to assess the diagnostic performance of serum hTERT in CCA. The result demonstrated that serum hTERT could discriminate between CCA patients and healthy individuals with high diagnostic sensitivity and speci city. Additional to CCA, the predictive function of hTERT was also determined in other malignancies. In gastric cancer, the expression pro le of hTERT showed close link with clinical factors and disease-free survival of the patients that might be a potential prognostic biomarker for the cancer [30]. The plasma hTERT mRNA level was signi cantly different between healthy individuals and prostate cancer cases, and ROC analysis demonstrated that hTERT was an useful non-invasive diagnostic biomarker for the disease [31]. Taken together, hTERT might be a potential molecular biomarker for human cancer that might be widely used for early detection, targeted treatment, and prognosis evaluation in tumor.

Conclusion
Page 7/12 In conclusion, serum hTERT expression is signi cantly increased in CCA patients, and positively related with aggressive clinical characteristics. Serum hTERT may be a potential biomarker for CCA diagnosis. The subjects had been informed the objective. Certainly, written consents were signed by every subject in this study.

Consent for publication
We obtaining permission from participants to publish their data.
Availability of data and materialsThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Competing interestsThe authors declare that they have no competing interests.  Relative expression value of serum hTERT mRNA in CCA patients and healthy individuals. The level of serum hTERT mRNA in CCA showed signi cantly higher than that in healthy controls (***: indicated P<0.001).